Rna polymerase variants

ABSTRACT

The present disclosure provides, in some aspects, variant RNA polymerases, the use of which increases transcription efficiency while reducing the number of double-stranded RNA contaminates and run-on transcripts produced during an in vitro transcription reaction.

RELATED APPLICATIONS

This application is a continuation of international application numberPCT/US2018/046989, filed Aug. 17, 2018, which was published under PCTArticle 21(2) in English and claims the benefit under 35 U.S.C. § 119(e)of U.S. provisional application No. 62/547,677, filed Aug. 18, 2017,U.S. provisional application No. 62/628,484, filed Feb. 9, 2018, U.S.provisional application No. 62/638,684, filed Mar. 5, 2018, and U.S.provisional application No. 62/677,527, filed May 29, 2018, each ofwhich is incorporated by reference herein in its entirety.

BACKGROUND

In vitro transcription (IVT) uses bacteriophage DNA-dependentribonucleic acid (RNA) polymerases (e.g., SP6, T3 and T7) to synthesizetemplate-directed mRNA transcripts. Problems in the IVT reaction canresult in complete failure (e.g., no transcript generated) or intranscripts that are the incorrect size (e.g., shorter or longer thanexpected). Specific problems associated with IVT reactions include, forexample, abortive (truncated) transcripts, run-on transcripts, polyAtail variants/3′ heterogeneity, mutated transcripts, and/ordouble-stranded contaminants produced during the reactions.

RNA polymerases exhibit three phases of transcription—initiation,elongation and termination. During the initiation phase, the RNApolymerase binds to a specific promoter DNA sequence, opens the DNAduplex and feeds the template strand into the active site. T7 RNApolymerase, for example, forms a structure referred to as initiationcomplex, which includes a six-helix bundle sub-domain (the promoterbinding domain) that interacts with the promoter to initiate DNA duplexmelting. While bound to the promoter, the polymerase produces many short(truncated) transcripts from 2-12 nucleotides (nt) in length, a processoften referred to as abortive synthesis/initiation. The truncated RNAtranscripts cannot be converted to full-length transcripts by RNApolymerase and become by-products that accumulate during transcription.After the transition to the elongation phase and release of thepromoter, the polymerase proceeds down the DNA template producing afull-length RNA transcript.

During the elongation phase, RNA polymerase often continues totranscribe DNA beyond the point at which termination should beinitiated, generating longer than expected RNA transcripts (“run-ontranscripts”). T7 RNA polymerase, for example, adds nucleotides to theend of a transcript before ‘falling off’ the template. Studies suggestthat more than 70% of transcripts generated by T7 RNA polymerase invitro may be run-on transcripts. In some cases, these aberrant RNAproducts are twice the length of the encoded sequence. Because run-ontranscription is stochastic, there is often great 3′ heterogeneity amongproducts in a given IVT reaction. This 3′ heterogeneity is problematicfor downstream applications, such as ligation reactions, which aredependent on RNA transcripts of a defined length and/or nucleotidecomposition.

SUMMARY

During the initiation of transcription, RNA polymerase balances twoopposing phases. The polymerase must first associate with the promotertightly enough to permit dissociation of the two DNA strands (one ofwhich is the template strand) and commence transcription. The polymerasemust then release the promoter and enter a highly processive elongationphase. Competition between these two phases leads to the production ofabortive transcripts. The polymerase repeatedly tries to clear thepromoter but is unable to overcome the transition barrier and releasesshort (abortive) RNA products. Conversely, the polymerase often fails to‘run-off’ the DNA template, generating a population of RNA transcriptswith 3′ heterogeneity. Provided herein are variant RNA polymerases,which increase transcription efficiency and 3′ homogeneity, run-ontranscripts, double-stranded contaminants, or any combination thereof,produced during an in vitro transcription (IVT) reaction, for example.

During the transition from initiation to elongation, RNA polymeraseundergoes a conformational change requiring a large rearrangement of theamino-terminal domain (N-terminal domain) (see, e.g., Bandwar, R P etal. Journal of Biological Chemistry 282, 22879-22886 (2009); Guillerez,J. et al. Proc National Acad Sci 102, 5958-5963 (2005); Durniak, K. etal. Science (New York, N.Y.)322, 553 (2008); and Tahirov, T. H. et al.Nature 420, 43-50 (2002), each of which is incorporated herein byreference. Within this N-terminal domain is a “C-helix” (e.g., aminoacids 28-71 of T7 RNA polymerase) and a “C-linker” (e.g., amino acids258-266 of T7 RNA polymerase), each of which contains subregions (aminoacids 42-47 and 257-262, respectively) that undergo a conformationalchange from a loop structure to a helix structure as the RNA polymerasetransitions from an initiation complex to an elongation complex (see,e.g., FIG. 10), abolishing the promoter binding site, enlarging theactive site and creating an exit tunnel for the RNA transcript.Mutations to subregions in the C-helix structure and/or the C-linkerlinker region may drive the conformation equilibrium toward theelongation complex by increasing the thermodynamic stability of theelongation complex relative to the initiation complex. Without beingbound by theory, it is also thought that mutations in select regions,such as the C-helix structure and/or the C-linker linker region, canchange how the exit tunnel of the polymerase interacts with thetranscript during elongation, causing a significant reduction intranscription errors (e.g., run-on transcripts). Thus, the variantpolymerases of the present disclosure include a (at least one) mutationin the C-helix and/or C-linker to drive the conformation equilibriumtoward the elongation complex.

Other regions of the N-terminal domain that contain loop structures thattransition to helix structures as the polymerase proceeds frominitiation to elongation may be mutated (point mutation, referred to asa substitution), as provided herein. Non-limiting examples of suchloop-to-helix regions include regions that span amino acids 55-73,164-169, or 176-187 of T7 RNA polymerase (e.g., SEQ ID NO:1), or regionsof other single subunit RNA polymerases (see, e.g., Cermakian, N. et al.J Mol Evol 45, 671-681 (1997), incorporated herein by reference) thatare homologous (e.g., at least 80%, at least 90%, at least 95%, or atleast 98% identical to) to the foregoing regions. Non-limiting examplesof other single subunit RNA polymerases include T3 RNA polymerase, K11RNA polymerase, and SP6 RNA polymerase.

In some embodiments, the RNA polymerase variants (e.g. T7 RNA polymerasevariants, include mutations to residues that have a high-helixpropensity (e.g., alanine) or that constrain the backbone flexibility tospecifically match that of the elongation complex.

Further provided herein, are RNA polymerase variants that include atleast one additional amino acid in the C terminus of the polymerase. Forexample, T7 RNA polymerase variants may include at least one additionalamino acid, such as an additional glycine (G) in the C terminus of thepolymerase. Surprisingly, the population of RNA transcripts producedusing a T7 RNA polymerase modified to include an additional G in theC-terminal “foot” region (e.g., the region comprising “FAFA” (SEQ ID NO:172) amino acids at positions 880-883 of wild-type T7 RNAP) exhibitsless 3′ heterogeneity. For example, as shown in FIG. 23, a T7 RNApolymerase variant that includes a C-terminal glycine (i.e., . . . FAFAG(SEQ ID NO: 329)) produces a RNA transcript population in which at least85% of the transcripts are homogeneous at the 3′ end. This data isparticularly unexpected, given that previous studies have shown thatC-terminal additions/insertions resulted in loss of function of T7polymerase (Gardner L P, et al. Biochemistry 36, 2908-2918 (1997) andGross L, et al. Journal of Molecular Biology 228, 488-505 (1992)).

Also provided herein are methods of capping a ssRNA (e.g., mRNA) withcap analogs (e.g., trinucleotides) co-transcriptionally(co-transcriptional capping methods) in an in vitro transcription assayusing a T7 RNA polymerase variant described herein (e.g., T7 RNApolymerase variant G47A or G47A* C-terminal variant). Efficientco-transcriptional capping typically includes a double-stranded DNA(dsDNA) template that initiates transcription with 5′ATP and equimolarconcentrations of NTPs and trinucleotides. Under these conditions, T7RNA polymerase exhibits severely reduced initiation activity with 5′ATP.Unexpectedly, the data provided herein shows that, in the presence ofGAG trinucleotides (e.g., m⁷GpppA_(2′OMe)pG), for example, the limitedinitiation activity of T7 RNA polymerase with 5′ATP drives initiationwith the trinucleotide rather than 5′ATP, generating capped RNA productsco-transcriptionally. Surprisingly, in some embodiments, greater than90% of the RNA produced comprises single-stranded full-lengthtranscripts, at least 90% of the RNA produced includes a functional cap,and the RNA produced does not elicit a substantial cytokine response,even in the absence of post-IVT purification.

Thus, some aspects of the present disclosure provide RNA polymerasevariants comprising at least one amino acid substitution, relative towild-type polymerase, that causes at least one loop structure of the RNApolymerase variant to undergo a conformational change to a helixstructure as the RNA polymerase variant transitions from an initiationcomplex to an elongation complex. In some embodiments, at least oneamino acid substitution is estimated to have a more negative change infolding free energy in the elongation complex than in the initiationcomplex. In some embodiments, at least one amino acid substitution has ahigh-helix propensity, relative to wild-type amino acid. In someembodiments, the RNA polymerase variant comprises an (at least one)additional amino acid residue at the C terminus. For example, an RNApolymerase variant (e.g., a T7 RNA polymerase variant) may comprise aglycine (G) at the C terminus.

In some embodiments, the RNA polymerase is a T7 RNA polymerase. In someembodiments, the RNA polymerase is a T3 RNA polymerase. In someembodiments, the RNA polymerase is a SP6 RNA polymerase. In someembodiments, the RNA polymerase is a K11 RNA polymerase.

In some embodiments, at least one loop structure is in the C-helixstructure. In some embodiments, at least one loop structure is in theC-linker structure. In some embodiments, at least one loop structure ispresent in a region within amino acids 55-73, 164-169, or 176-187 of T7RNA polymerase, or a RNA polymerase homologous to T7 RNA polymerase.

In some embodiments, at least one amino acid substitution is at leastone high-helix propensity amino acid substitution. For example, at leastone high-helix propensity amino acid substitution may be selected fromalanine, isoleucine, leucine, arginine, methionine, lysine, glutamine,and/or glutamate. In some embodiments, at least one high-helixpropensity amino acid substitution is alanine. In some embodiments, atleast one high-helix propensity amino acid substitution is isoleucine.In some embodiments, at least one high-helix propensity amino acidsubstitution is leucine. In some embodiments, at least one high-helixpropensity amino acid substitution is arginine. In some embodiments, atleast one high-helix propensity amino acid substitution is methionine.In some embodiments, at least one high-helix propensity amino acidsubstitution is lysine. In some embodiments, at least one high-helixpropensity amino acid substitution is glutamine. In some embodiments, atleast one high-helix propensity amino acid substitution is glutamate.

In some embodiment, a T7 RNA polymerase is modified to include at leastone amino acid substitution of a high-helix propensity amino acid in atleast one position selected from E42 (e.g., E42R), S43 (e.g., S43A), Y44(e.g., Y44A), E45 (e.g., E45R/L), M46 (e.g., M46A), G47 (e.g., G47A),A255 (e.g., A255K/Q/Y/I), R257 (e.g., R257A), A258 (e.g., A258R/E/L),G259 (e.g., G259A), A260 (e.g., A260R/E/L), L261 (e.g., L261A) and A262(e.g., A262R/E/L). The T7 RNA polymerase may further comprise, in someembodiments, one or more additional amino acid substitutions (inaddition to at least one high-helix propensity amino acid substitution).Thus, the present disclosure encompasses the further modification ofexisting (e.g., currently-available and/or commercially-available) T7RNA polymerase variants with one or more high-helix propensity aminoacid substitutions as provided herein.

In some embodiments, a T7 RNA polymerase comprises an amino acidsequence of SEQ ID NO:1 modified to include at least one amino acidsubstitution of a high-helix propensity amino acid at a positionselected from E42 (e.g., E42R), S43 (e.g., S43A), Y44 (e.g., Y44A), E45(e.g., E45R/L), M46 (e.g., M46A) and G47 (e.g., G47A). In someembodiments, at least one amino acid substitution comprises S43A. Insome embodiments, at least one amino acid substitution comprises G47A.

In some embodiments, a T7 RNA polymerase comprises an amino acidsequence of SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:294, SEQ ID NO:295,or SEQ ID NO:296 modified to include at least one amino acidsubstitution of a high-helix propensity amino acid at a positionselected from E42 (e.g., E42R), S43 (e.g., S43A), Y44 (e.g., Y44A), E45(e.g., E45R/L), M46 (e.g., M46A) and G47 (e.g., G47A). In someembodiments, at least one amino acid substitution comprises S43A. Insome embodiments, at least one amino acid substitution comprises G47A.

In some embodiments, a T7 RNA polymerase comprises an amino acidsequence of SEQ ID NO:1 modified to include at least one amino acidsubstitution of a high-helix propensity amino acid at a positionselected from A255 (e.g., A255K/Q/Y/I), R257 (e.g., R257A), A258 (e.g.,A258R/E/L), G259 (e.g., G259A), A260 (e.g., A260R/E/L), L261 (e.g.,L261A) and A262 (e.g., A262R/E/L). In some embodiments, at least oneamino acid substitution comprises R257A. In some embodiments, at leastone amino acid substitution comprises G259A.

In some embodiments, a T7 RNA polymerase comprises an amino acidsequence of SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:294, SEQ ID NO:295,or SEQ ID NO:296 modified to include at least one amino acidsubstitution of a high-helix propensity amino acid at a positionselected from A255 (e.g., A255K/Q/Y/I), R257 (e.g., R257A), A258 (e.g.,A258R/E/L), G259 (e.g., G259A), A260 (e.g., A260R/E/L), L261 (e.g.,L261A) and A262 (e.g., A262R/E/L). In some embodiments, at least oneamino acid substitution comprises R257A. In some embodiments, at leastone amino acid substitution comprises G259A.

Also provided herein, in some aspects, are T7 RNA polymerases comprisingan amino acid sequence of SEQ ID NO:1 modified to include an amino acidsubstitution of a high-helix propensity amino acid (e.g., alanine,isoleucine, leucine, arginine, methionine, lysine, glutamine, and/orglutamate) at position G47, S43, R257, or G259. Further provided herein,in some aspects, are T7 RNA polymerases comprising an amino acidsequence of SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:294, SEQ ID NO:295,or SEQ ID NO:296 modified to include an amino acid substitution of ahigh-helix propensity amino acid (e.g., alanine, isoleucine, leucine,arginine, methionine, lysine, glutamine, and/or glutamate) at positionG47, S43, R257, or G259.

In some embodiments, T7 RNA polymerases of the present disclosurecomprise an amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4or SEQ ID NO:5. In some embodiments, T7 RNA polymerases of the presentdisclosure comprise an amino acid sequence of SEQ ID NO:107 or 108, SEQID NO:109 or 110, SEQ ID NO:111 or 112, or SEQ ID NO:113 or 114.

In some embodiments, a T7 RNA polymerase comprises at least oneadditional C-terminal amino acid. In some embodiments, a T7 RNApolymerase comprises at least two additional C-terminal amino acids.

In some embodiments, the at least two additional C-terminal amino acidscomprise the same type of amino acid (e.g., all Gly, all Ala). In someembodiments, the at least two additional C-terminal amino acids compriseat least two different types of amino acids (e.g., GlyAla, AlaGly).

In some embodiments, a T7 RNA polymerase comprises at least threeadditional C-terminal amino acids. In some embodiments, the at leastthree additional C-terminal amino acids comprise at least two or atleast three of the same type or different types of amino acids (e.g.,GlyGlyGly, AlaAlaAla).

In some embodiments, a T7 RNA polymerase comprises 1 to 10 (e.g., 1, 2,3, 4, 5, 6, 7, 8, 9, or 10) additional C-terminal amino acids. In someembodiments, a T7 RNA polymerase comprises 1 to 5 additional C-terminalamino acids.

In some embodiments, a T7 RNA polymerase comprises a C terminus thatcomprises a FAFAX_(n) (SEQ ID NO: 171) motif, wherein X is any aminoacid and n is any integer greater than zero. In some embodiments, X isglycine (G). In some embodiments, n is 1, 2, 3, 4, or 5. It should beunderstood that in embodiments, where n is greater than 1, such that theC terminal motif is FAFXX, FAFAXXX (SEQ ID NO: 319), FAFAXXXX (SEQ IDNO: 320), or FAFAXXXXX (SEQ ID NO: 321), for example, the X's may be thesame amino acid or they may be different amino acids. For example, theC-terminal motif may be FAFAGG (SEQ ID NO: 322) or FAFAGGG (SEQ ID NO:323), or the C-terminal motif may be FAFAGA (SEQ ID NO: 324), FAFAGC(SEQ ID NO: 325), FAFAGAA (SEQ ID NO: 326), FAFAGAG (SEQ ID NO: 327),FAFAGAC (SEQ ID NO: 328), etc. Other C-terminal amino acid combinationsmay be used.

In some embodiments, a T7 RNA polymerase comprises a C terminus thatcomprises a FAFAG (SEQ ID NO: 329) motif.

In some embodiments, a T7 RNA polymerase comprises a XAFAX_(n) motif, aFXFAX_(n) motif, FAXAX_(n) motif, or a FAFXX_(n) motif, wherein each Xis any amino acid and n is any integer greater than zero. Thus, thepresent disclosure comprises various C-terminal F⁸⁸⁰A⁸⁸¹F⁸⁸²A⁸⁸³ (SEQ IDNO: 172) motifs, wherein the amino acid at one or more of positions 880,881, 882, or 883 (e.g., relative to wild-type T7 RNAP, e.g., SEQ IDNO:1) is modified to include at least one amino acid substitution, withor without an additional C-terminal amino acid (X_(n)).

In some aspects, the present disclosure provides RNA polymerasescomprising at least one additional C-terminal amino acid relative to acorresponding wild-type RNA polymerase. In some embodiments, the RNApolymerase is selected from T7 RNA polymerases, T3 RNA polymerases, andSP6 RNA polymerases.

In some embodiments, the RNA polymerase further comprises at least oneadditional amino acid substitution. In some embodiments, the RNApolymerase further comprises an amino acid substitution corresponding toan amino acid substitution of SEQ ID NO:1 selected from G47A, S43A,R257A, and G259A.

In some embodiments, the RNA polymerase is a T7 RNA polymerasecomprising an amino acid sequence at least 90%, at least 95%, at least98%, or at least 99% identical to SEQ ID NO:1 modified to include anamino acid substitution at position 43±1 (e.g., 42, 43, or 44), 47±1(e.g., 46, 47, or 48), 257±1 (e.g., 256, 257, or 258), and/or 259±1(e.g., 258, 259, or 260), optionally wherein the amino acid substitutionis alanine (A). In some embodiments, the RNA polymerase is a T3 RNApolymerase comprising an amino acid sequence at least 90%, at least 95%,or at least 98% identical to SEQ ID NO:6 modified to include an aminoacid substitution at a position corresponding to wild-type T7 RNApolymerase position 43±1, 47±1, 257±1, and/or 259±1 (based on a sequenceor structural alignment), optionally wherein the amino acid substitutionis alanine (A). In some embodiments, the RNA polymerase is a SP6 RNApolymerase comprising an amino acid sequence at least 90%, at least 95%,at least 98%, or at least 99% identical to SEQ ID NO:7 modified toinclude an amino acid substitution at a position corresponding towild-type T7 RNA polymerase position 43±1, 47±1, 257±1, and/or 259±1(based on a sequence or structural alignment), optionally wherein theamino acid substitution is alanine (A).

Some aspects of the present disclosure provide T7 RNA polymerasescomprising an amino acid sequence at least 90%, at least 95%, at least98%, or at least 99% identical to SEQ ID NO:1 modified to include anamino acid substitution at position 43, 47, 257, and/or 259, optionallywherein the amino acid substitution is alanine (A).

Other aspects of the present disclosure provide T3 RNA polymerasescomprising an amino acid sequence at least 90%, at least 95%, or atleast 98% identical to SEQ ID NO:6 modified to include an amino acidsubstitution at a position corresponding to wild-type T7 RNA polymeraseposition 43, 47, 257, and/or 259, optionally wherein the amino acidsubstitution is alanine (A). Thus, in some embodiments, T3 RNApolymerases comprise an amino acid sequence at least 90%, at least 95%,or at least 98% identical to SEQ ID NO:6 modified to include an aminoacid substitution at position 44, 48, 258, and/or 260, optionallywherein the amino acid substitution is alanine (A).

Yet other aspects of the present disclosure provide SP6 RNA polymerasescomprising an amino acid sequence at least 90%, at least 95%, or atleast 98% % identical to SEQ ID NO:7 modified to include an amino acidsubstitution at a position corresponding to wild-type T7 RNA polymeraseposition 43, 47, 257, and/or 259, optionally wherein the amino acidsubstitution is alanine (A). Thus, in some embodiments, SP6 TNApolymerases comprise an amino acid sequence at least 90%, at least 95%,or at least 98% % identical to SEQ ID NO:7 modified to include an aminoacid substitution at position 15, 19, 230, and/or 232, optionallywherein the amino acid substitution is alanine (A).

The present disclosure also provides methods of producing RNA comprisingcontacting a DNA template with a RNA polymerase variant as describedherein under conditions that result in the production of RNA transcript(e.g., under IVT condition).

The present disclosure further provides methods of performing an IVTreaction, comprising contacting a DNA template with a RNA polymerasevariant as provided herein in the presence of nucleoside triphosphates(NTPs) and buffer under conditions that result in the production of RNAtranscripts.

In some embodiments, the RNA transcript produced, when delivered tocells, optionally in unpurified form, stimulates cytokine response thatis at least 50% (e.g., at least 60%, at least 70%, at least 80%, atleast 95% or at least 98%) lower relative to RNA produced usingwild-type RNA polymerase under the same IVT conditions.

In some embodiments, the concentration of dsRNA transcript produced byIVT is at least 50% (e.g., at least 60%, at least 70%, at least 80%, atleast 95% or at least 98%) lower relative to dsRNA transcript producedusing wild-type polymerase.

In some embodiments, less than 20% (e.g., less than 15%, less than 10%,less than 5%) of the RNA transcripts produced exhibit 3′ heterogeneity.

In some embodiments, less than 50% (e.g., less than 40%, less than 30%,less than 20%, less than 10%) of the RNA transcript produced istruncated RNA transcript.

In some embodiments, less than 50% (e.g., less than 40%, less than 30%,less than 20%, less than 10%) of the RNA transcript produced is run-onRNA transcript.

In some embodiments, the amount of full-length RNA transcript producedis at least 15 times greater than the amount of the DNA template

In some embodiments, the ratio of truncated RNA transcript:full-lengthRNA transcript produced is less than 1:1.

In some embodiments, the RNA transcript produced has less than 1mutation per 100 nucleotides relative to the DNA template.

The present disclosure, in some aspects provides nucleic acids encodingthe RNA polymerase variants and, in some embodiments, vectors (e.g.,plasmids) and/or host cells (e.g., mammalian cells, e.g., human cells)comprising the nucleic acids.

The RNA transcripts produced by the methods of the present disclosureare also provided. In some embodiments, the RNA transcripts (e.g., mRNA)are formulated in a lipid nanoparticle. The lipid nanoparticle maycomprise, for example, a molar ratio of 20-60% ionizable amino lipid,5-25% non-cationic lipid, 25-55% sterol, and 0.5-15% PEG-modified lipid.See, e.g., WO 2017/070624, published 27 Apr. 2017, incorporated hereinby reference.

Other compositions and kits comprising the RNA polymerase variants areencompassed herein.

Also provided herein are co-transcriptional capping methods forribonucleic acid (RNA) synthesis, the methods comprising reacting apolynucleotide template with a T7 RNA polymerase variant, nucleosidetriphosphates, and a cap analog under in vitro transcription reactionconditions to produce RNA transcript.

In some embodiments, greater than 80%, greater than 85%, or greater than90% of the RNA transcript produced includes a functional cap. In someembodiments, greater than 95% of the RNA transcript produced includes afunctional cap.

In some embodiments, the nucleoside triphosphates comprise unmodified ormodified ATP, modified or unmodified UTP, modified or unmodified GTP,and/or modified or unmodified CTP.

In some embodiments, the T7 polymerase variant comprises an amino acidsequence of SEQ ID NO:1 (or an amino acid sequence that shares 90%-99%,e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% identity with SEQ ID NO:1) modified to include atleast one amino acid substitution of a high-propensity amino acid at aposition selected from E42, S43, Y44, E45, M46, G47, A255, R257, andG259. In some embodiments, the T7 polymerase variant comprises an aminoacid sequence of SEQ ID NO:1 (or an amino acid sequence that shares90%-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%,at least 98%, or at least 99% identity with SEQ ID NO:1) modified toinclude amino acid substitution of G47A. In some embodiments, the T7polymerase variant comprises an amino acid sequence of SEQ ID NO:1 (oran amino acid sequence that shares 90%-99%, e.g., at least 90%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% identitywith SEQ ID NO:1) modified to include amino acid substitution of S43A.

In some embodiments, the T7 polymerase variant comprises an amino acidsequence of SEQ ID NO:99 (or an amino acid sequence that shares 90%-99%,e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% identity with SEQ ID NO:99) modified to include atleast one amino acid substitution of a high-propensity amino acid at aposition selected from E42, S43, Y44, E45, M46, G47, A255, R257, andG259. In some embodiments, the T7 polymerase variant comprises an aminoacid sequence of SEQ ID NO:99 (or an amino acid sequence that shares90%-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%,at least 98%, or at least 99% identity with SEQ ID NO:99) modified toinclude amino acid substitution of G47A. In some embodiments, the T7polymerase variant comprises an amino acid sequence of SEQ ID NO:99 (oran amino acid sequence that shares 90%-99%, e.g., at least 90%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% identitywith SEQ ID NO:99) modified to include amino acid substitution of S43A.

In some embodiments, the T7 polymerase variant comprises an amino acidsequence of SEQ ID NO:100 (or an amino acid sequence that shares90%-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%,at least 98%, or at least 99% identity with SEQ ID NO:100) modified toinclude at least one amino acid substitution of a high-propensity aminoacid at a position selected from E42, S43, Y44, E45, M46, G47, A255,R257, and G259. In some embodiments, the T7 polymerase variant comprisesan amino acid sequence of SEQ ID NO:100 (or an amino acid sequence thatshares 90%-99%, e.g., at least 90%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identity with SEQ ID NO:100) modifiedto include amino acid substitution of G47A. In some embodiments, the T7polymerase variant comprises an amino acid sequence of SEQ ID NO:100 (oran amino acid sequence that shares 90%-99%, e.g., at least 90%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% identitywith SEQ ID NO:100) modified to include amino acid substitution of S43A.

In some embodiments, the nucleoside triphosphates and cap analog arepresent in the reaction at equimolar concentrations. In someembodiments, a molar ratio of cap analog to nucleoside triphosphates inthe reaction is greater than 1:1. In some embodiments, a molar ratio ofcap analog to nucleoside triphosphates in the reaction is less than 1:1.

In some embodiments, the cap analog is a dinucleotide cap, atrinucleotide cap, or a tetranucleotide cap. In some embodiments, thecap analog is a trinucleotide cap.

In some embodiments, the trinucleotide cap comprises a sequence selectedfrom the following sequences: GAA, GAC, GAG, GAU, GCA, GCC, GCG, GCU,GGA, GGC, GGG, GGU, GUA, GUC, GUG, and GUU.

In some embodiments, the trinucleotide cap comprises a sequence selectedfrom the following sequences: m⁷GpppApA, m⁷GpppApC, m⁷GpppApG,m⁷GpppApU, m⁷GpppCpA, m⁷GpppCpC, m⁷GpppCpG, m⁷GpppCpU, m⁷GpppGpA,m⁷GpppGpC, m⁷GpppGpG, m⁷GpppGpU, m⁷GpppUpA, m⁷GpppUpC, m⁷GpppUpG, andm⁷GpppUpU.

In some embodiments, the trinucleotide cap comprises a sequence selectedfrom the following sequences: m⁷G_(3′OMe)pppApA, m⁷G_(3′OMe)pppApC,m⁷G_(3′OMe)pppApG, m⁷G_(3′OMe)pppApU, m⁷G_(3′OMe)pppCpA,m⁷G_(3′OMe)pppCpC, m⁷G_(3′OMe)pppCpG, m⁷G_(3′OMe)pppCpU,m⁷G_(3′OMe)pppGpA, m⁷G_(3′OMe)pppGpC, m⁷G_(3′OMe)pppGpG,m⁷G_(3′OMe)pppGpU, m⁷G_(3′OMe)pppUpA, m⁷G_(3′OMe)pppUpC,m⁷G_(3′OMe)pppUpG, and m⁷G_(3′OMe)pppUpU.

In some embodiments, the trinucleotide cap comprises a sequence selectedfrom the following sequences: m⁷G_(3′OMe)pppA_(2′OMe)pA,m⁷G_(3′OMe)pppA_(2′OMe)pC, m⁷G_(3′OMe)pppA_(2′OMe)pG,m⁷G_(3′OMe)pppA_(2′OMe)pU, m⁷G_(3′OMe)pppC_(2′OMe)pA,m⁷G_(3′OMe)pppC_(2′OMe)pC, m⁷G_(3′OMe)pppC_(2′OMe)pG,m⁷G_(3′OMe)pppC_(2′OMe)pU, m⁷G_(3′OMe)pppG_(2′OMe)pA,m⁷G_(3′OMe)pppG_(2′OMe)pC, m⁷G_(3′OMe)pppG_(2′OMe)pG,m⁷G_(3′OMe)pppG_(2′OMe)pU, m⁷G_(3′OMe)pppU_(2′OMe)pA,m⁷G_(3′OMe)pppU_(2′OMe)pC, m⁷G_(3′OMe)pppU_(2′OMe)pG, andm⁷G_(3′OMe)pppU_(2′OMe)pU.

In some embodiments, the trinucleotide cap comprises a sequence selectedfrom the following sequences: m⁷GpppA_(2′OMe)pA, m⁷GpppA_(2′OMe)pC,m⁷GpppA_(2′OMe)pG, m⁷GpppA_(2′OMe)pU, m⁷GpppC_(2′OMe)pA,m⁷GpppC_(2′OMe)pC, m⁷GpppC_(2′OMe)pG, m⁷GpppC_(2′OMe)pU,m⁷GpppG_(2′OMe)pA, m⁷GpppG_(2′OMe)pC, m⁷GpppG_(2′OMe)pG,m⁷GpppG_(2′OMe)pU, m⁷GpppU_(2′OMe)pA, m⁷GpppU_(2′OMe)pC,m⁷GpppU_(2′OMe)pG, and m⁷GpppU_(2′OMe)pU.

In some embodiments, the trinucleotide cap comprises a sequence selectedfrom the following sequences: GAG, GCG, GUG, and GGG. In someembodiments, the trinucleotide cap comprises sequence GAG. In someembodiments, the trinucleotide cap comprises m⁷GpppA_(2′Ome)pG.

In some embodiments, the polynucleotide template includes a2′-deoxythymidine residue at template position +1. In some embodiments,the polynucleotide template includes a 2′-deoxycytidine residue attemplate position +1. In some embodiments, the polynucleotide templateincludes a 2′-deoxyadenosine residue at template position +1. In someembodiments, the polynucleotide template includes a 2′-deoxyguanosineresidue at template position +1.

Also provided herein are co-transcriptional capping methods for RNAsynthesis, the methods comprising reacting a polynucleotide templatewith (a) a T7 RNA polymerase variant comprising at least one amino acidsubstitution, relative to wild-type RNA polymerase, that causes at leastone loop structure of the RNA polymerase variant to undergo aconformational change to a helix structure as the RNA polymerase varianttransitions from an initiation complex to an elongation complex, (b)nucleoside triphosphates, and (c) a trinucleotide cap comprisingsequence GpppA_(2′Ome)pG, under in vitro transcription reactionconditions to produce RNA transcript, wherein the polynucleotidetemplate includes a 2′-deoxythymidine residue at template position +1.

In some embodiments, the RNA transcript produced, when delivered tocells, optionally in unpurified form, does not stimulate a detectablecytokine response.

Further provided herein are compositions comprising an invitro-transcribed (IVT) RNA and a pharmaceutically acceptable excipient,wherein the composition is substantially free of cytokine-inducing RNAcontaminant in the absence of post-IVT purification.

A composition, in some embodiments, comprises an IVT RNA and apharmaceutically acceptable excipient, wherein the composition has lessthan 5% uncapped RNA species.

In some embodiments, greater than 80%, 85%, or 90% of the IVT RNAinclude a functional cap. In some embodiments, greater than 95% of theIVT RNA include a functional cap.

In some embodiments, the IVT RNA is not chemically modified. In otherembodiments, the IVT RNA is chemically modified.

In some embodiments, greater than 95% of the IVT RNA comprisessingle-stranded full-length transcripts.

In some embodiments, the RNA is produced by a process comprising:reacting a polynucleotide template with (a) a T7 RNA polymerase variantcomprising at least one amino acid substitution, relative to wild-typeRNA polymerase, that causes at least one loop structure of the RNApolymerase variant to undergo a conformational change to a helixstructure as the RNA polymerase variant transitions from an initiationcomplex to an elongation complex, (b) nucleoside triphosphates, and (c)a trinucleotide cap comprising sequence GpppA_(2′Ome)pG, under in vitrotranscription reaction conditions to produce RNA transcript, wherein thepolynucleotide template includes a 2′-deoxythymidine residue at templateposition +1.

In some aspects, the disclosure provides T7 RNAP variants comprising anamino acid sequence of SEQ ID NOs: 294-313, wherein x is any amino acidand n is any integer, e.g., between 1 and 5 (e.g., 1, 2, 3, 4, or 5).

In some aspects, the disclosure provides a method of performing an IVTreaction, comprising contacting a DNA template with an RNA polymerasevariant of the disclosure in the presence of nucleoside triphosphate andbuffer under conditions that result in the production of RNAtranscripts. In some embodiments, the RNA produced, when delivered tocells, optionally in unpurified form, stimulates a cytokine responsethat is at least 50% lower relative to the dsRNA transcript producedusing a WT. In some embodiments, less than 30% of the RNA transcriptsproduced by the IVT reaction exhibit 3′ heterogeneity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows HPLC chromatograms at 260 nm of human erythropoietin (hEPO)mRNA generated using wild-type (WT) T7 polymerase or T7 polymerasevariants, G47A* (with C-terminal G) and S43A* (with C-terminal G).

FIG. 2, left panel, shows a graph depicting IFNβ response in BJfibroblasts transfected with chemically-unmodified hEPO RNA transcriptproduced using WT T7 RNA polymerase or one of the T7 RNA polymerasevariants, S43A* (with C-terminal G) or G47A* (with C-terminal G), withor without reverse phase (RP) purification. FIG. 2, right panel, shows agraph depicting hEPO expression in transfected cells.

FIG. 3, top panel, shows a graph depicting IFNβ response in BJfibroblasts transfected with chemically-modified hEPO RNA transcripts(N1-methylpseudouracil (m¹ψ) modifications) produced using WT T7 RNApolymerase or one of the T7 RNA polymerase variants, S43A* (withC-terminal G) or G47A* (with C-terminal G), with or without reversephase (RP) purification. FIG. 3, bottom panel, shows a graph depictingIFNβ response in cells transfected with chemically-modified hEPO RNAtranscripts (5-methoxy-uridine (mo⁵U) modifications) produced using WTT7 RNA polymerase or one of the T7 RNA polymerase variants, S43A* (withC-terminal G) or G47A* (with C-terminal G), with or without reversephase (RP) purification.

FIG. 4, top and bottom panels, show graphs depicting hEPO expression incells transfected with the hEPO RNA transcripts used for FIG. 3.N1-methylpseudouracil (m¹ψ) modified hEPO expression is shown in the topgraph, and 5-methoxy-uridine (mo⁵U) modified hEPO expression is shown inthe bottom graph.

FIG. 5 shows a graph of IP10 response in monocyte-derived macrophagestransfected with chemically-unmodified hEPO RNA transcript producedusing WT T7 RNA polymerase or T7 RNA polymerase variants, S43A* (withC-terminal G) or G47A* (with C-terminal G).

FIG. 6, top panel, shows a graph of IP10 response in monocyte-derivedmacrophages transfected with chemically-modified (N1-methylpseudouracil(m¹ψ)) hEPO RNA transcript produced using WT T7 RNA polymerase or T7 RNApolymerase variants, S43A* (with C-terminal G) or G47A* (with C-terminalG). FIG. 6, bottom panel, shows a graph of IP10 response inmonocyte-derived macrophages transfected with chemically-modified(5-methoxy-uridine (mo⁵U)) hEPO RNA transcript produced using WT T7 RNApolymerase or T7 RNA polymerase variants, S43A* (with C-terminal G) orG47A* (with C-terminal G).

FIG. 7 shows graphs depicting concentration of contaminatingdouble-stranded (ds) RNA detected using a dsRNA ELISA with 1 μg ofchemically-unmodified hEPO RNA transcript (left) or 5 μgchemically-unmodified hEPO RNA transcript produced via IVT reactionusing WT T7 RNA polymerase or T7 RNA polymerase variants, S43A* (withC-terminal G) or G47A* (with C-terminal G).

FIG. 8 shows graphs depicting concentration of contaminating dsRNAdetected using a dsRNA ELISA with chemically-modified hEPO RNAtranscript (N1-methylpseudouracil (m¹ψ) modification, top;5-methoxy-uridine (mo⁵U) modification, bottom).

FIGS. 9A-9B show mass chromatogram results from a RNase T1 tail digestof hEPO RNA transcript produced using WT T7 RNA polymerase of T7 RNApolymerase variants, S43A* (with C-terminal G) or G47A* (with C-terminalG). FIG. 9A shows results from a LCMS analysis. FIG. 9B showsquantification of the 3′ end population distribution from FIG. 9A.

FIG. 10 shows a schematic of the C-helix and C-linker loop structureschanging conformation into C-helix and C-loop helix structures as the T7RNA polymerase transitions from the initiation complex to the elongationcomplex. FIG. 10 was generated from PDB crystal structures 1MSW and 1QLNand rendered in Molecular Operating Environment [Chemical ComputingGroup ULC].

FIG. 11A shows a schematic of using a DNA splint in the present ofligase to ligate a “leftmer” RNA transcript produced by IVT to a“rightmer” fluorescently-labeled polyA signal. Even a single nucleotideoverhang at the ligation site abolishes ligation efficiently.

FIG. 11B is a PAGE-D gel showing ligation efficiency for rightmersproduced with WT T7 RNA polymerase and T7 RNA polymerase variants, G47A*(with C-terminal G) or S43A* (with C-terminal G).

FIG. 12 shows a radioactive gel using 32P-GTP to label abortivetranscripts or 32P-CTP to label reverse complements. The IVTs wereperformed using a short model transcript. This data demonstrates that T7RNA polymerase variants S43A* (with C-terminal G) and G47A* (withC-terminal G) reduce reverse complement formation.

FIG. 13 is a schematic of a convention method of producing a capped mRNAvia in vitro transcription.

FIGS. 14A-14E are graphs showing results of a co-transcriptional cappingassay using either wild-type T7 RNA polymerase or a T7 RNA polymerasevariant of the present disclosure (G47A*). FIG. 14A shows examples ofdinucleotide- (Vaccinia cap1) or trinucleotide- (GAG or GmAG) caps.Trinucleotide caps were used in the co-transcriptional capping assay.FIG. 14B shows the mRNA yields of the co-transcriptional capping assay.

FIG. 14C shows that the mRNAs produced in the co-transcriptional cappingassay were of high integrity. FIG. 14D shows the capped mRNAs producedby WT T7 RNA polymerase induced cytokine production in BJ fibroblasts,while the capped mRNAs produced by T7 RNA polymerase variant G47A* (withC-terminal G), surprisingly, did not induce cytokine production. FIG.14E shows the capped mRNAs produced by WT T7 RNA polymerase did notexpress the encoded protein (hEPO) in BJ fibroblasts, while the cappedmRNAs produced by T7 RNA polymerase variant G47A* (with C-terminal G)lead to hEPO expression level comparable to the control. The control isa mRNA produced by WT T7 RNA polymerase and capped with Vaccinia cap1.

FIG. 15 shows the results of LC-mass spectrometry analysis of the mRNAproduced in the co-transcriptional capping assay. The result showsunexpectedly that T7 RNA polymerase variant G47A* (with C-terminal G)produced cleaner mRNA than WT T7 RNA polymerase. The trinucleotideincorporation rates appeared equivalent for both enzymes.

FIGS. 16A and 16B are graphs comparing the cytokine response (FIG. 16A)or expression (FIG. 16B) of mRNAs produced and capped with GAGtrinucleotide in the co-transcriptional capping assay or capped in astandard capping assay with Vaccinia cap1.

FIG. 17 is a schematic showing the co-transcriptionally capping assaydescribed herein using T7 RNA polymerase variant G47A* and atrinucleotide cap m⁷GpppA_(2′Ome)pG.

FIGS. 18A and 18B are liquid chromatography-mass spectrometry (LCMS)results showing the comparison of capping efficiency for mRNAs thatstart with 5′ATP or 5′GTP. FIG. 18A shows the analysis of intact mRNAs.FIG. 18B shows the analysis of the 5′ ends of the mRNAs cleaved by RNaseH.

FIGS. 19A-19C are graphs showing the analysis of mRNA chemicallymodified with 1-methyl-pseudouridine produced from PCR fragmenttemplates for three model constructs (hEPO, luciferase, and eGFP). FIG.19A shows that the mRNAs have the same sequence, as analyzed by RNase T1fingerprinting assay. FIG. 19B shows that the mRNAs have high degree ofintegrity. FIG. 19C shows that the mRNAs did not induce cytokineresponse in BJ fibroblasts.

FIGS. 20A-20F are graphs showing the analysis of mRNAs chemicallymodified with 1-methyl-pseudouridine produced from plasmid templates forthree model constructs (hEPO, Luc, and eGFP). The NTP consumption in theco-transcriptional capping assays using hEPO plasmids (FIG. 20A) or eGFPplasmids (FIG. 20B) as templates are shown. FIG. 20C shows that the mRNAproducts are of high integrity. FIG. 20D shows the cytokine response ofthe mRNA products. FIG. 20E shows the expression of mRNA encoding hEPOin BJ fibroblasts. FIG. 20F shows the expression of mRNA encodingluciferase (Luc) in BJ fibroblasts. FIG. 20G shows the expression ofmRNA encoding eGFP in BJ fibroblasts.

FIG. 21 is a graph showing that greater than 85% (e.g., ˜90%) of themRNA transcripts produced using T7 RNAP variant G47A* comprising aC-terminal glycine (G) have a hydroxyl group at the 3′ end.

FIG. 22 shows unmodified hEPO mRNA production from IVT reactionscontaining WT or G47A T7 RNAP variants, some of which have additionalamino acids at the C-terminus (e.g., one or two glycines, or twoalanines).

FIG. 23 shows 1-methyl-pseudouridine-modified hEPO mRNA production fromIVT reactions containing WT, G47A, or S43A/G47A T7 RNAP variants asindicated, some of which have additional amino acids at the C-terminus(e.g., one or two glycines or two alanines).

FIGS. 24A-24D show results from a RNase T1 tail digest of hEPO mRNAtranscript produced using WT, G47A, or S43A/G47A T7 RNAP variants asindicated, some of which have additional amino acids at the C-terminus(e.g., one or two glycines or two alanines). FIG. 24A shows results froma LCMS analysis of hEPO mRNA produced from IVT reactions using WT T7RNAP with equimolar concentrations of NTPs (WT EQ), WT T7 RNAP withexcess of GTP and ATP (WT alpha), or G47A* T7 RNAP with an additionalC-terminal glycine with equimolar concentrations of NTPs (G47A* EQ).FIG. 24B shows quantification of the 3′ end population distribution fromFIG. 24A. FIG. 24C shows the percentage of clean 3′ end populationsproduced in IVT reactions using unmodified mRNA. FIG. 24D shows thepercentage of clean 3′ end populations produced in IVT reactions using1-methyl-pseudouridine-modified mRNA.

FIGS. 25A-25B are graphs comparing the cytokine response of BJfibroblasts to unmodified (FIG. 25A) and 1-methyl-pseudouridine-modified(FIG. 25B) mRNAs produced from IVT reactions containing WT, G47A, orS43A/G47A T7 RNAP variants as indicated, some of which havesubstitutions at the C-terminus.

FIGS. 26A-26B show radiolabeled mRNAs produced from IVT reactionscontaining WT or G47A T7 RNAP variants as indicated, some of which haveadditional amino acids at the C-terminus. The IVT mRNA products areseparated by size on a polyacrylamide denaturing gel. In FIG. 26A,either unmodified or 1-methyl-pseudouridine-modified mRNA isradiolabeled with ³²P-CTP. In FIG. 26B, the reverse complement mRNA isradiolabeled with ³²P-CTP.

FIGS. 27A and 27B show graphs of data demonstrating that trinuc-cappedG47A* mRNAs encoding firefly luciferase (ffLuc) induce IP-10 serumcytokine levels in vivo at Day 1 (FIG. 27A) and Day 29 (FIG. 27B) thatare similar to mRNA controls.

FIGS. 28A and 28B show graphs of data demonstrating that trinuc-cappedG47A* mRNAs encoding ffLuc induce baseline cytokine levels in vitro inBJ fibroblasts (BJFs) (FIG. 28A) and in monocyte-derived macrophages(MDMs) (FIG. 28B) that are similar to mRNA controls.

FIG. 29 shows a graph of data demonstrating that trinuc-capped G47A*mRNAs encoding ffLuc maintain high expression in vivo after six (6)weekly doses.

FIG. 30 shows a graph of data demonstrating that trinuc-capped G47A*mRNAs encoding ffLuc induce low anti-PEG IgM levels after six (6) weeklydoses.

FIG. 31 shows a graph of data demonstrating that trinuc-capped G47A*mRNAs encoding ffLuc exhibit low B cell activation, similar to mRNAcontrols.

FIGS. 32A and 32B show graphs of data demonstrating that trinuc-cappedG47A* mRNAs encoding human erythropoietin (hEPO) induce IP-10 serumcytokine levels in vivo at Day 1 (FIG. 32A) and Day 22 (FIG. 32B) thatare similar to mRNA controls.

FIGS. 33A and 33B show graphs of data demonstrating that trinuc-cappedG47A* mRNAs encoding hEPO induce baseline cytokine levels in vitro in BJfibroblasts (BJFs) (FIG. 33A) and in monocyte-derived macrophages (MDMs)(FIG. 33B) that are similar to mRNA controls.

FIG. 34 shows a graph of data demonstrating that trinuc-capped G47A*mRNAs encoding hEPO maintain high expression in vivo after six (6)weekly doses.

FIG. 35 shows a graph of data demonstrating that trinuc-capped G47A*mRNAs encoding hEPO induce low anti-PEG IgM levels after six (6) weeklydoses.

FIG. 36 shows a graph of data demonstrating that trinuc-capped G47A*mRNAs encoding hEPO exhibit low B cell activation, similar to mRNAcontrols.

FIGS. 37A and 37B show that the G47A* T7 RNA polymerase variant does notaffect indel frequency (FIG. 37A) or point mutation frequency (FIG.37B).

DETAILED DESCRIPTION

The present disclosure provides RNA polymerase (RNAP) variants thatincrease transcription efficiency and 3′ homogeneity while reducing thenumber of run-on transcripts, and/or double-stranded contaminantsproduced during an in vitro transcription (IVT) reaction, for example.These RNAP variants, which include, in some embodiments, a single aminoacid substitution, facilitate the RNAP conformational transition frominitiation complex to elongation complex, thereby reducing many of theproblems associated with the transcription initiation phase.

Provided herein are unexpected experimental results demonstrating thatmodification(s) of the N-terminal C-helix and/or C-linker structure(s)of DNA-dependent RNA polymerase (e.g., T7 RNA polymerase) drives theconformation equilibrium of the polymerase toward the elongationcomplex, facilitating release of the DNA template promoter andcommencement of a highly processive elongation phase. Surprisingly, useof the polymerase variants provided herein in IVT reactions reduces 3′heterogeneity among the transcripts produced, and also reduces (oreliminates) the production of double-stranded contaminants. Further,results show that the purity and expression levels of transcriptsproduced using the polymerase variants are comparable to those oftranscripts produced using wild-type polymerase.

Thus, use of the RNA polymerase variants of the present disclosurereduces many of the problems associated with IVT reaction products.Although wild-type T7 RNA polymerase (RNAP) is used commonly in bothindustry and academia, several of its activities significantlycompromise the purity of resulting RNA transcripts. In particular, useof this wild-type T7 RNAP enzyme results in the non-templated additionof nucleotides to the 3′-end of RNA transcripts. For example, wild-typeT7 RNAP installs at least 1, and often 2 or more, non-templatednucleotides at the 3′-end with seemingly little preference fornucleobase identity. Surprisingly, the T7 RNAP variants as providedherein reduce the occurrence of 3′ heterogeneity. In some embodiments,less than 30% of the RNA transcripts produced by IVT using a RNApolymerase variant of the present disclosure exhibit 3′ heterogeneity.In some embodiments, less than 20% (e.g., less than 15%, 10%, or 5%) ofthe RNA transcripts produced by IVT using a RNA polymerase variant ofthe present disclosure exhibit 3′ heterogeneity. In some embodiments,1-20%, 1-15%, 1-10%, or 1-5% of the RNA transcripts produced by IVTexhibit 3′ heterogeneity. In some embodiments, less than 1% of the RNAtranscripts produced by IVT using a RNA polymerase variant of thepresent disclosure exhibit 3′ heterogeneity.

Thus, use of the RNA polymerase variants of the present disclosure, forexample, in an IVT reaction, results in the production of ‘cleaner’ RNAtranscript (a population of RNA transcripts with reducedheterogeneity/increased homogeneity at the 3′ end). In some embodiments,at least 70% of the RNA transcripts produced using the RNA polymerasevariants of the present disclosure exhibit 3′ homogeneity. In someembodiments, at least 75%, at least 80%, at least 85%, at least 90%, orat least 95% of the RNA transcripts produced using the RNA polymerasevariants of the present disclosure exhibit 3′ homogeneity. In someembodiments, at least 90% of the RNA transcripts produced using the RNApolymerase variants of the present disclosure exhibit 3′ homogeneity. Insome embodiments, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96% at least 97%, at least98%, or at least 99% of the RNA transcripts produced using the RNApolymerase variants of the present disclosure exhibit 3′ homogeneity.

Also, surprising herein were results demonstrating that the RNAtranscripts produced using the amino acid substitution (e.g., S43Aand/or G47A of SEQ ID NO:1) or C-terminal additions (e.g., WT, S43A*,and/or G47A* with one or more amino acid additions) T7 RNA polymerasevariants, when delivered to cells, optionally in unpurified form (e.g.,not purified by reverse phase chromatography), stimulates a cytokineresponse that is lower relative to RNA produced using wild-type RNApolymerase. In some embodiments, there is no detectable cytokineresponse from cells that have received an RNA transcript produced usinga T7 RNA variant polymerase of the present disclosure. In someembodiments, RNA produced using a RNA polymerase variant (e.g., T7 RNAPS43A* variant and/or T7 RNAP G47A* variant) stimulates a cytokineresponse that is at least 50% (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95% or 100%) lower relative to RNA produced using wild-type RNApolymerase. In some embodiments, RNA produced using a RNA polymerasevariant (e.g., T7 RNAP S43A* variant and/or T7 RNAP G47A* variant)stimulates a cytokine response that is 50-60%, 50-70%, 50-80%, 50-90%,50-100%, 60-70%, 60-80%, 60-90%, 60-100%, 70-80%, 70-90%, 70-100%,80-90%, 80-100% or 90-100% lower relative to RNA produced usingwild-type RNA polymerase. In some embodiments, RNA produced using a RNApolymerase variant (e.g., T7 RNAP S43A* variant and/or T7 RNAP G47A*variant) stimulates a cytokine response that is at least 2-fold, 3-fold,4-fold or 5-fold lower relative to RNA produced using wild-type RNApolymerase. In some embodiments, the cells used to test a cytokineresponse are human fibroblast cells (e.g., BJ (ATCC® CRL-2522™) cells).In some embodiments, the cells used to test a cytokine response aremonocyte-derived macrophages (MDMs).

In some embodiments, the concentration of dsRNA transcript producedusing a RNA polymerase variant is at least 50% (e.g., 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95% or 100%) lower relative to dsRNA transcriptproduced using wild-type polymerase. In some embodiments, theconcentration of dsRNA transcript produced is 50-60%, 50-70%, 50-80%,50-90%, 50-100%, 60-70%, 60-80%, 60-90%, 60-100%, 70-80%, 70-90%,70-100%, 80-90%, 80-100% or 90-100% lower relative to dsRNA transcriptproduced using wild-type polymerase. In some embodiments, theconcentration of dsRNA transcript produced is at least 2-fold, 3-fold,4-fold or 5-fold lower relative to dsRNA transcript produced usingwild-type polymerase.

Use of the RNA polymerase variants of the present disclosure alsoresulted unexpectedly in the production of double stranded contaminantsand fewer run-on transcripts.

In some embodiments, less than 50% (e.g., less than 45%, 40%, 35%, 30%,25%, 20%, 15%, 10%, 5% or 1%) of the RNA transcript produced, e.g., byIVT, using a RNA polymerase variant is double-stranded contaminant. Insome embodiments, 1-50%, 1-40%, 1-30%, 1-20%, 1-10%, 1-5%, 5-50%, 5-40%,5-30%, 5-20%, 5-10%, 10-50%, 10-40%, 10-30% or 10-20% of the RNAtranscript produced is double-stranded contaminant.

In some embodiments, less than 50% (e.g., less than 45%, 40%, 35%, 30%,25%, 20%, 15%, 10%, 5% or 1%) of the RNA transcript produced using a RNApolymerase variant is run-on RNA transcript. In some embodiments, 1-50%,1-40%, 1-30%, 1-20%, 1-10%, 1-5%, 5-50%, 5-40%, 5-30%, 5-20%, 5-10%,10-50%, 10-40%, 10-30% or 10-20% of the RNA transcript produced isrun-on RNA transcript.

In some embodiments, the amount of full-length RNA transcript producedusing a RNA polymerase variant is at least 15 times greater than theamount of the DNA template. For example, the amount of full-length RNAtranscript produced may be at least 20, 30, 40, 45, 50, 60, 70, 80, 90or 100 times greater than the amount of the DNA template. In someembodiments, the amount of full-length RNA transcript produced is15-100, 15-90, 15-80, 15-70, 15-60, 15-50, 15-40, 15-30, 15-20, 20-100,20-90, 20-80, 20-70, 20-60, 20-50, 20-40 or 20-30 times greater than theamount of the DNA template. In some embodiments, the amount offull-length RNA transcript produced is 2-fold, 3-fold, 4-fold or 5-foldgreater than the amount of the DNA template.

In some embodiments, the ratio of double-strandedcontaminant:full-length RNA transcript produced using a RNA polymerasevariant is less than 1:1. For example, the ratio of double-strandedcontaminant:full-length RNA transcript produced may be 0.9:1, 0.8:1,0.7:1, 0.6:1, 0.5:1, 0.4:1, 0.3:1, 0.2:1 or 0.1:1.

In some embodiments, the RNA transcript produced using a RNA polymerasevariant has less than 1 mutation per 100 nucleotides relative to the DNAtemplate. For example, the RNA transcript produced may have less than 1mutation per 200, 300, 400, 500, 600, 700, 800, 900 or 1000 nucleotidesrelative to the DNA template.

RNA Polymerases

RNA polymerase (DNA-dependent RNA polymerase) is an enzyme thatcatalyzes the sequential addition of a ribonucleotide to the 3′ end of agrowing RNA chain (transcription of RNA in the 5′→3′ direction), withnucleoside triphosphates (NTPs) acting as substrates for the enzyme andwith the sequence of nucleotides specified by a DNA template.Transcription relies on the complementary pairing of bases. The twostrands of a double helix separate locally, and one of the separatedstrands serves as a template (DNA template). RNA polymerase thencatalyzes the alignment of free nucleotides on the DNA template by theircomplementary bases in the template. Thus, a RNA polymerase isconsidered to have RNA polymerase activity if the polymerase catalyzesthe sequential addition of a ribonucleotide to the 3′ end of a growingRNA chain.

T7 RNA polymerase (T7 RNAP) is a 99 kDa DNA-dependent RNA polymeraseencoded by the genome of bacteriophage T7 and is highly specific for T7phage promoters. Structural studies of T7 RNAP have shown that theconformation of the N-terminal domain changes substantially between theinitiation phase and elongation phase of transcription. The N-terminaldomain comprises a C-helix subdomain and the promoter binding domain,which includes two segments separated by subdomain H. The promoterbinding domain and the bound promoter rotate by approximately 45 degreesupon synthesis of an 8-nt RNA transcript, allowing the promoter contactsto be maintained while the active site is expanded to accommodate agrowing heteroduplex. The C-helix subdomain moves modestly toward itselongation conformation, whereas subdomain H remains in itsinitiation—rather than its elongation-phase location, more than 70angstroms away. Comparison of the structures of the T7 RNAP initiationand elongation complexes reveal extensive conformational changes withinthe N-terminal 267 residues (N-terminal domain) and little change in therest of the RNAP. A rigid body rotation of the promoter binding domainas well as the refolding of the N-terminal C-helix (residues 28-71) andH (residues 151-190) subdomains are responsible for abolishing thepromoter binding site, enlarging the active site and creating an exittunnel for the RNA transcript. The structural changes within theN-terminal domain account for the increased stability and theprocessivity of the elongation complex (see, e.g., Durniak, K. J. etal., Science 322(5901): 553-557, 2008, incorporated herein byreference).

Provided herein, in some aspects, are RNA polymerase variants (e.g., T7RNAP variants) that facilitate the conformational change from the RNAPinitiation complex to the RNAP elongation complex. A RNA polymerasevariant is an enzyme having RNA polymerase activity and at least onesubstitution relative to the counterpart wild-type RNA polymerase. Asindicated above, a RNA polymerase is considered to have RNA polymeraseactivity if the polymerase catalyzes the sequential addition of aribonucleotide to the 3′ end of a growing RNA chain. For example, anenzyme that includes the amino acid sequence SEQ ID NO:1 with an aminoacid substitution at position S43 (e.g., S43A) or G47 (e.g., G47A) andmaintains RNA polymerase activity is considered a T7 RNAP variant ofwild-type T7 RNAP (SEQ ID NO:1).

In some embodiments, a RNA polymerase variant comprises at least oneamino acid modification, relative to wild-type RNA polymerase, thatcauses at least one three-dimensional loop structure of the RNApolymerase variant to undergo a conformational change to a helixstructure as the RNA polymerase variant transitions from an initiationcomplex to an elongation complex. Thus, in some embodiments, at leastone amino acid modification has a high-helix propensity, relative towild-type amino acid.

Examples of loop structures include but are not limited to amino acid(aa) 42-47 in the C-helix structure (e.g., aa 28-71 of SEQ ID NO:1) ofthe T7 RNA polymerase initiation complex (IC) conformation and aa257-262 in the C-linker structure (e.g., aa 258-266 of SEQ ID NO:1) ofthe IC.

Also provided herein are RNA polymerase variants (e.g., T7 RNAPvariants) that include at least one additional amino acid at the Cterminus. The at least one additional amino acid, in some embodiments,is selected from alanine, arginine, asparagine, aspartic acid, cysteine,glutamic acid, glutamine, glycine, histidine, isoleucine, leucine,lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine. In some embodiments, the at least oneadditional amino acid is a polar amino acid. In some embodiments, the atleast one additional amino acid is a non-polar amino acid. In someembodiments, the at least one additional amino acid is glycine. In someembodiments, the at least one additional amino acid is alanine. In someembodiments, the at least one additional amino acid is serine.

In some embodiments, the C terminus of an RNA polymerase comprises thefollowing consensus sequence: FAFAX_(n) (SEQ ID NO:171), wherein x isany amino acid and n is any integer, e.g., between 1 and 5 (e.g., 1, 2,3, 4, or 5). In some embodiments, the C terminus of an RNA polymerasecomprises the following consensus sequence: FAFAG_(n) (SEQ ID NO:330),wherein n is any integer, e.g., between 1 and 5. In some embodiments,the C terminus of an RNA polymerase comprises the following consensussequence: FAFAA_(n) (SEQ ID NO:331), wherein n is any integer, e.g.,between 1 and 5. In some embodiments, the C terminus of an RNApolymerase comprises the following consensus sequence: FAFAR_(n) (SEQ IDNO:332), wherein n is any integer, e.g., between 1 and 5. In someembodiments, the C terminus of an RNA polymerase comprises the followingconsensus sequence: FAFAN_(n) (SEQ ID NO:333), wherein n is any integer,e.g., between 1 and 5. In some embodiments, the C terminus of an RNApolymerase comprises the following consensus sequence: FAFAD_(n) (SEQ IDNO:334), wherein n is any integer, e.g., between 1 and 5. In someembodiments, the C terminus of an RNA polymerase comprises the followingconsensus sequence: FAFAC_(n) (SEQ ID NO:335), wherein n is any integer,e.g., between 1 and 5. In some embodiments, the C terminus of an RNApolymerase comprises the following consensus sequence: FAFAE_(n) (SEQ IDNO:336), wherein n is any integer, e.g., between 1 and 5. In someembodiments, the C terminus of an RNA polymerase comprises the followingconsensus sequence: FAFAQ_(n) (SEQ ID NO:302), wherein n is any integer,e.g., between 1 and 5. In some embodiments, the C terminus of an RNApolymerase comprises the following consensus sequence: FAFAH_(n) (SEQ IDNO:303), wherein n is any integer, e.g., between 1 and 5. In someembodiments, the C terminus of an RNA polymerase comprises the followingconsensus sequence: FAFAI_(n) (SEQ ID NO:304), wherein n is any integer,e.g., between 1 and 5. In some embodiments, the C terminus of an RNApolymerase comprises the following consensus sequence: FAFAL_(n) (SEQ IDNO:305), n is any integer, e.g., between 1 and 5. In some embodiments,the C terminus of an RNA polymerase comprises the following consensussequence: FAFAK_(n) (SEQ ID NO:306), wherein n is any integer, e.g.,between 1 and 5. In some embodiments, the C terminus of an RNApolymerase comprises the following consensus sequence: FAFAM_(n) (SEQ IDNO:307), wherein n is any integer, e.g., between 1 and 5. In someembodiments, the C terminus of an RNA polymerase comprises the followingconsensus sequence: FAFAF_(n) (SEQ ID NO:308), wherein n is any integer,e.g., between 1 and 5. In some embodiments, the C terminus of an RNApolymerase comprises the following consensus sequence: FAFAP_(n) (SEQ IDNO:309), wherein n is any integer, e.g., between 1 and 5. In someembodiments, the C terminus of an RNA polymerase comprises the followingconsensus sequence: FAFAS_(n) (SEQ ID NO:310), wherein n is any integer,e.g., between 1 and 5. In some embodiments, the C terminus of an RNApolymerase comprises the following consensus sequence: FAFAT_(n) (SEQ IDNO:311), wherein n is any integer, e.g., between 1 and 5. In someembodiments, the C terminus of an RNA polymerase comprises the followingconsensus sequence: FAFAW_(n) (SEQ ID NO:312), wherein n is any integer,e.g., between 1 and 5. In some embodiments, the C terminus of an RNApolymerase comprises the following consensus sequence: FAFAY_(n) (SEQ IDNO:313), n is any integer, e.g., between 1 and 5. In some embodiments,the C terminus of an RNA polymerase comprises the following consensussequence: FAFAV_(n) (SEQ ID NO:314), wherein n is any integer, e.g.,between 1 and 5.

In some embodiments, the C-terminal motif (FAFA (SEQ ID NO: 172) orFAFAX_(n) (SEQ ID NO: 171)) comprises an amino acid substitution at oneor more of positions 880, 881, 882, or 883, relative to wild-type T7RNAP (e.g., SEQ ID NO:1). Thus, the present disclosure comprises variousC-terminal F⁸⁸⁰A⁸⁸¹F⁸⁸²A⁸⁸³ (SEQ ID NO: 172) motifs, wherein the aminoacid at one or more of positions 880, 881, 882, or 883 relative towild-type T7 RNAP (e.g., SEQ ID NO:1) is modified to include at leastone amino acid substitution, with or without an additional C-terminalamino acid (X_(n)).

Amino Acid Substitutions

RNA polymerase variants of the present disclosure include at least oneamino acid substitution, relative to the WT RNA polymerase. For example,with reference to WT T7 RNA polymerase having an amino acid sequence ofSEQ ID NO:1, the serine at position 43 is considered a “wild-type aminoacid,” whereas a substitution of the serine for alanine at position 43is considered an “amino acid substitution” that has a high-helixpropensity.

The average globular protein contains 30% α-helix, the most common typeof secondary structure. Some amino acids occur more frequently inα-helices than others; this tendency is known as helix propensity. See,e.g., Pace, N.C. and Scholtz, J. M. Biophysical Journal, 75:422-427(1998), incorporated herein by reference. In some embodiments, at leastone amino acid substitution has a high-helix propensity, relative towild-type amino acid. In general, high-helix propensity amino acidsubstitutions are selected to thermodynamically bias the population ofpolymerase conformers toward the elongation complex. The relative MGs(free energies) is calculated for substitutions in the IC and theelongation complex (EC) structures using publicly-available software(e.g., University of Washington's Rosetta and Schrödinger's Maestro).Substitutions are then selected based on the calculations and onadditional knowledge, including for example, amino acid helix propensityand/or polypeptide backbone phi-psi compatibility.

In some embodiments, the RNA polymerase variant is a T7 RNA polymerasevariant comprising at least one (one or more) amino acid substitutionrelative to WT T7 RNA polymerase (e.g., WT T7 RNA polymerase having anamino acid sequence of SEQ ID NO:1). In some embodiments, an amino acidsubstitution is a high-helix propensity amino acid substitution.Examples of high-helix propensity amino acids include alanine,isoleucine, leucine, arginine, methionine, lysine, glutamine, and/orglutamate.

In some embodiments, the RNA polymerase variant is a T7 RNA polymerasevariant comprising at least one additional amino acid at the C terminus(e.g., SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:294, SEQ ID NO:295, or SEQID NO:296, or an amino acid sequence that shares 90%-99%, e.g., at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% identity with SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:294, SEQ IDNO:295, or SEQ ID NO:296). In some embodiments, the RNA polymerasevariant is a T7 RNA polymerase variant comprising at least oneadditional amino acid at the C terminus and an amino acid substitutionrelative to WT T7 RNA polymerase. In some embodiments, the amino acidsubstitution is a high-helix propensity amino acid substitution.

Provided herein are two approaches that may be used to identify high orhigher helix propensity amino acid substitutions that thermodynamicallybias/favor the conformational equilibrium toward the EC. In bothapproaches, care is taken to avoid making amino acid substitutions toany positions that are directly involved in promoter binding orcatalysis (based on review of the structures, literature searches). InMethod A, specialized software is used to compute the change in freeenergy of protein folding due to mutation (ΔΔG_(mut)) in the EC and IC.All possible amino acid substitutions at the desired sequence positionsare evaluated by this method. Desired amino acid substitutions (i.e.,those that favor the EC over the IC) are those for which the ΔΔG_(mut)difference (ΔΔG_(mut) ^(EC)−ΔΔG_(mut) ^(IC)) is negative. In Method B,the IC and EC secondary structure annotations (e.g., as determined from3D models of IC and EC tertiary structures using the DSSP software(Dictionary of protein secondary structure: pattern recognition ofhydrogen-bonded and geometrical features. Kabsch W, Sander C,Biopolymers. 1983 22 2577-2637)) are inspected to identify regions witha loop in the IC and a helix in the EC. Sequence positions with lowhelix propensity residues (e.g., Gly) are then substituted with highhelix propensity amino acids (e.g., Ala). For T7 RNAP, for example,mutational (e.g., substitutional) overlap from Methods A and B wereidentified as well as the top variant from Method B.

In some embodiments, the amino acid substitution is a high-helixpropensity amino acid (e.g., alanine, isoleucine, leucine, arginine,methionine, lysine, glutamine, and/or glutamate) substitution at any oneof amino acid positions 42-47 (E42, S43, Y44, E45, M46 and/or G47) ofSEQ ID NO:1. In some embodiments, the amino acid substitution is ahigh-helix propensity amino acid (e.g., alanine, isoleucine, leucine,arginine, methionine, lysine, glutamine, and/or glutamate) substitutionat any one of amino acid positions 42-47 (E42, S43, Y44, E45, M46 and/orG47) of an amino acid sequence that shares 90-99%, e.g., at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with SEQ ID NO:1. In some embodiments, the amino acidsubstitution is a high-helix propensity amino acid substitution at aminoacid position 42 of SEQ ID NO:1. In some embodiments, the amino acidsubstitution is a high-helix propensity amino acid substitution at aminoacid position 43 of SEQ ID NO:1. In some embodiments, the amino acidsubstitution is a high-helix propensity amino acid substitution at aminoacid position 44 of SEQ ID NO:1. In some embodiments, the amino acidsubstitution is a high-helix propensity amino acid substitution at aminoacid position 45 of SEQ ID NO:1. In some embodiments, the amino acidsubstitution is a high-helix propensity amino acid substitution at aminoacid position 46 of SEQ ID NO:1. In some embodiments, the amino acidsubstitution is a high-helix propensity amino acid substitution at aminoacid position 47 of SEQ ID NO:1.

In some embodiments, the amino acid substitution is a high-helixpropensity amino acid (e.g., alanine, isoleucine, leucine, arginine,methionine, lysine, glutamine, and/or glutamate) substitution at any oneof amino acid positions 42-47 (E42, S43, Y44, E45, M46 and/or G47) ofSEQ ID NO:99, 100, 294, 295, or 296. In some embodiments, the amino acidsubstitution is a high-helix propensity amino acid (e.g., alanine,isoleucine, leucine, arginine, methionine, lysine, glutamine, and/orglutamate) substitution at any one of amino acid positions 42-47 (E42,S43, Y44, E45, M46 and/or G47) of an amino acid sequence that shares90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identity with SEQ ID NO:99, 100, 294, 295, or296. In some embodiments, the amino acid substitution is a high-helixpropensity amino acid substitution at amino acid position 42 of SEQ IDNO:99, 100, 294, 295, or 296. In some embodiments, the amino acidsubstitution is a high-helix propensity amino acid substitution at aminoacid position 43 of SEQ ID NO:99, 100, 294, 295, or 296. In someembodiments, the amino acid substitution is a high-helix propensityamino acid substitution at amino acid position 44 of SEQ ID NO:99, 100,294, 295, or 296. In some embodiments, the amino acid substitution is ahigh-helix propensity amino acid substitution at amino acid position 45of SEQ ID NO:99, 100, 294, 295, or 296. In some embodiments, the aminoacid substitution is a high-helix propensity amino acid substitution atamino acid position 46 of SEQ ID NO:99, 100, 294, 295, or 296. In someembodiments, the amino acid substitution is a high-helix propensityamino acid substitution at amino acid position 47 of SEQ ID NO:99, 100,294, 295, or 296.

In some embodiments, the amino acid substitution is an alanine at anyone of amino acid positions 42-47 of SEQ ID NO:1. In some embodiments,the amino acid substitution is an alanine at any one of amino acidpositions 42-47 of an amino acid sequence that shares 90-99%, e.g., atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identity with SEQ ID NO:1. Thus, in some embodiments, theamino acid substitution is an alanine at position 42 of SEQ ID NO:1. Insome embodiments, the amino acid substitution is an alanine at position43 of SEQ ID NO:1. In some embodiments, the amino acid substitution isan alanine at position 44 of SEQ ID NO:1. In some embodiments, the aminoacid substitution is an alanine at position 45 of SEQ ID NO:1. In someembodiments, the amino acid substitution is an alanine at position 46 ofSEQ ID NO:1. In some embodiments, the amino acid substitution is analanine at position 47 of SEQ ID NO:1.

In some embodiments, the amino acid substitution is an alanine at anyone of amino acid positions 42-47 of SEQ ID NO:99, 100, 294, 295, or296. In some embodiments, the amino acid substitution is an alanine atany one of amino acid positions 42-47 of an amino acid sequence thatshares 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identity with SEQ ID NO:99, 100, 294,295, or 296. Thus, in some embodiments, the amino acid substitution isan alanine at position 42 of SEQ ID NO:99, 100, 294, 295, or 296. Insome embodiments, the amino acid substitution is an alanine at position43 of SEQ ID NO:99, 100, 294, 295, or 296. In some embodiments, theamino acid substitution is an alanine at position 44 of SEQ ID NO:99,100, 294, 295, or 296. In some embodiments, the amino acid substitutionis an alanine at position 45 of SEQ ID NO:99, 100, 294, 295, or 296. Insome embodiments, the amino acid substitution is an alanine at position46 of SEQ ID NO:99, 100, 294, 295, or 296. In some embodiments, theamino acid substitution is an alanine at position 47 of SEQ ID NO:99,100, 294, 295, or 296.

In some embodiments, the amino acid substitution is an isoleucine at anyone of amino acid positions 42-47 of SEQ ID NO:1. In some embodiments,the amino acid substitution is an isoleucine at any one of amino acidpositions 42-47 of an amino acid sequence that shares 90-99%, e.g., atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identity with SEQ ID NO:1. Thus, in some embodiments, theamino acid substitution is an isoleucine at position 42 of SEQ ID NO:1.In some embodiments, the amino acid substitution is an isoleucine atposition 43 of SEQ ID NO:1. In some embodiments, the amino acidsubstitution is an isoleucine at position 44 of SEQ ID NO:1. In someembodiments, the amino acid substitution is an isoleucine at position 45of SEQ ID NO:1. In some embodiments, the amino acid substitution is anisoleucine at position 46 of SEQ ID NO:1. In some embodiments, the aminoacid substitution is an isoleucine at position 47 of SEQ ID NO:1.

In some embodiments, the amino acid substitution is an isoleucine at anyone of amino acid positions 42-47 of SEQ ID NO:99, 100, 294, 295, or296. In some embodiments, the amino acid substitution is an isoleucineat any one of amino acid positions 42-47 of an amino acid sequence thatshares 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identity with SEQ ID NO:99, 100, 294,295, or 296. Thus, in some embodiments, the amino acid substitution isan isoleucine at position 42 of SEQ ID NO:99, 100, 294, 295, or 296. Insome embodiments, the amino acid substitution is an isoleucine atposition 43 of SEQ ID NO:99, 100, 294, 295, or 296. In some embodiments,the amino acid substitution is an isoleucine at position 44 of SEQ IDNO:99, 100, 294, 295, or 296. In some embodiments, the amino acidsubstitution is an isoleucine at position 45 of SEQ ID NO:99, 100, 294,295, or 296. In some embodiments, the amino acid substitution is anisoleucine at position 46 of SEQ ID NO:99, 100, 294, 295, or 296. Insome embodiments, the amino acid substitution is an isoleucine atposition 47 of SEQ ID NO:99, 100, 294, 295, or 296.

In some embodiments, the amino acid substitution is a leucine at any oneof amino acid positions 42-47 of SEQ ID NO:1. In some embodiments, theamino acid substitution is a leucine at any one of amino acid positions42-47 of an amino acid sequence that shares 90-99%, e.g., at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with SEQ ID NO:1. Thus, in some embodiments, the amino acidsubstitution is a leucine at position 42 of SEQ ID NO:1. In someembodiments, the amino acid substitution is a leucine at position 43 ofSEQ ID NO:1. In some embodiments, the amino acid substitution is aleucine at position 44 of SEQ ID NO:1. In some embodiments, the aminoacid substitution is a leucine at position 45 of SEQ ID NO:1. In someembodiments, the amino acid substitution is a leucine at position 46 ofSEQ ID NO:1. In some embodiments, the amino acid substitution is aleucine at position 47 of SEQ ID NO:1.

In some embodiments, the amino acid substitution is a leucine at any oneof amino acid positions 42-47 of SEQ ID NO:99, 100, 294, 295, or 296. Insome embodiments, the amino acid substitution is a leucine at any one ofamino acid positions 42-47 of an amino acid sequence that shares 90-99%,e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% identity with SEQ ID NO:99, 100, 294, 295, or 296.Thus, in some embodiments, the amino acid substitution is a leucine atposition 42 of SEQ ID NO:99, 100, 294, 295, or 296. In some embodiments,the amino acid substitution is a leucine at position 43 of SEQ ID NO:99,100, 294, 295, or 296. In some embodiments, the amino acid substitutionis a leucine at position 44 of SEQ ID NO:99, 100, 294, 295, or 296. Insome embodiments, the amino acid substitution is a leucine at position45 of SEQ ID NO:99, 100, 294, 295, or 296. In some embodiments, theamino acid substitution is a leucine at position 46 of SEQ ID NO:99,100, 294, 295, or 296. In some embodiments, the amino acid substitutionis a leucine at position 47 of SEQ ID NO:99, 100, 294, 295, or 296.

In some embodiments, the amino acid substitution is an arginine at anyone of amino acid positions 42-47 of SEQ ID NO:1. In some embodiments,the amino acid substitution is an arginine at any one of amino acidpositions 42-47 of an amino acid sequence that shares 90-99%, e.g., atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identity with SEQ ID NO:1. Thus, in some embodiments, theamino acid substitution is an arginine at position 42 of SEQ ID NO:1. Insome embodiments, the amino acid substitution is an arginine at position43 of SEQ ID NO:1. In some embodiments, the amino acid substitution isan arginine at position 44 of SEQ ID NO:1. In some embodiments, theamino acid substitution is an arginine at position 45 of SEQ ID NO:1. Insome embodiments, the amino acid substitution is an arginine at position46 of SEQ ID NO:1. In some embodiments, the amino acid substitution isan arginine at position 47 of SEQ ID NO:1.

In some embodiments, the amino acid substitution is an arginine at anyone of amino acid positions 42-47 of SEQ ID NO:99, 100, 294, 295, or296. In some embodiments, the amino acid substitution is an arginine atany one of amino acid positions 42-47 of an amino acid sequence thatshares 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identity with SEQ ID NO:99, 100, 294,295, or 296. Thus, in some embodiments, the amino acid substitution isan arginine at position 42 of SEQ ID NO:99, 100, 294, 295, or 296. Insome embodiments, the amino acid substitution is an arginine at position43 of SEQ ID NO:99, 100, 294, 295, or 296. In some embodiments, theamino acid substitution is an arginine at position 44 of SEQ ID NO:99,100, 294, 295, or 296. In some embodiments, the amino acid substitutionis an arginine at position 45 of SEQ ID NO:99, 100, 294, 295, or 296. Insome embodiments, the amino acid substitution is an arginine at position46 of SEQ ID NO:99, 100, 294, 295, or 296. In some embodiments, theamino acid substitution is an arginine at position 47 of SEQ ID NO:99,100, 294, 295, or 296.

In some embodiments, the amino acid substitution is a methionine at anyone of amino acid positions 42-47 of SEQ ID NO:1. In some embodiments,the amino acid substitution is a methionine at any one of amino acidpositions 42-47 of an amino acid sequence that shares 90-99%, e.g., atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identity with SEQ ID NO:1. Thus, in some embodiments, theamino acid substitution is a methionine at position 42 of SEQ ID NO:1.In some embodiments, the amino acid substitution is a methionine atposition 43 of SEQ ID NO:1. In some embodiments, the amino acidsubstitution is a methionine at position 44 of SEQ ID NO:1. In someembodiments, the amino acid substitution is a methionine at position 45of SEQ ID NO:1. In some embodiments, the amino acid substitution is amethionine at position 46 of SEQ ID NO:1. In some embodiments, the aminoacid substitution is a methionine at position 47 of SEQ ID NO:1.

In some embodiments, the amino acid substitution is a methionine at anyone of amino acid positions 42-47 of SEQ ID NO:99, 100, 294, 295, or296. In some embodiments, the amino acid substitution is a methionine atany one of amino acid positions 42-47 of an amino acid sequence thatshares 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identity with SEQ ID NO:99, 100, 294,295, or 296. Thus, in some embodiments, the amino acid substitution is amethionine at position 42 of SEQ ID NO:99, 100, 294, 295, or 296. Insome embodiments, the amino acid substitution is a methionine atposition 43 of SEQ ID NO:99, 100, 294, 295, or 296. In some embodiments,the amino acid substitution is a methionine at position 44 of SEQ IDNO:99, 100, 294, 295, or 296. In some embodiments, the amino acidsubstitution is a methionine at position 45 of SEQ ID NO:99, 100, 294,295, or 296. In some embodiments, the amino acid substitution is amethionine at position 46 of SEQ ID NO:99, 100, 294, 295, or 296. Insome embodiments, the amino acid substitution is a methionine atposition 47 of SEQ ID NO:99, 100, 294, 295, or 296.

In some embodiments, the amino acid substitution is a lysine at any oneof amino acid positions 42-47 of SEQ ID NO:1. In some embodiments, theamino acid substitution is a lysine at any one of amino acid positions42-47 of an amino acid sequence that shares 90-99%, e.g., at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with SEQ ID NO:1. Thus, in some embodiments, the amino acidsubstitution is a lysine at position 42 of SEQ ID NO:1. In someembodiments, the amino acid substitution is a lysine at position 43 ofSEQ ID NO:1. In some embodiments, the amino acid substitution is alysine at position 44 of SEQ ID NO:1. In some embodiments, the aminoacid substitution is a lysine at position 45 of SEQ ID NO:1. In someembodiments, the amino acid substitution is a lysine at position 46 ofSEQ ID NO:1. In some embodiments, the amino acid substitution is alysine at position 47 of SEQ ID NO:1.

In some embodiments, the amino acid substitution is a lysine at any oneof amino acid positions 42-47 of SEQ ID NO:99, 100, 294, 295, or 296. Insome embodiments, the amino acid substitution is a lysine at any one ofamino acid positions 42-47 of an amino acid sequence that shares 90-99%,e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% identity with SEQ ID NO:99, 100, 294, 295, or 296.Thus, in some embodiments, the amino acid substitution is a lysine atposition 42 of SEQ ID NO:99, 100, 294, 295, or 296. In some embodiments,the amino acid substitution is a lysine at position 43 of SEQ ID NO:99,100, 294, 295, or 296. In some embodiments, the amino acid substitutionis a lysine at position 44 of SEQ ID NO:99, 100, 294, 295, or 296. Insome embodiments, the amino acid substitution is a lysine at position 45of SEQ ID NO:99, 100, 294, 295, or 296. In some embodiments, the aminoacid substitution is a lysine at position 46 of SEQ ID NO:99, 100, 294,295, or 296. In some embodiments, the amino acid substitution is alysine at position 47 of SEQ ID NO:99, 100, 294, 295, or 296.

In some embodiments, the amino acid substitution is a glutamine at anyone of amino acid positions 42-47 of SEQ ID NO:1. In some embodiments,the amino acid substitution is a glutamine at any one of amino acidpositions 42-47 of an amino acid sequence that shares 90-99%, e.g., atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identity with SEQ ID NO:1. Thus, in some embodiments, theamino acid substitution is a glutamine at position 42 of SEQ ID NO:1. Insome embodiments, the amino acid substitution is a glutamine at position43 of SEQ ID NO:1. In some embodiments, the amino acid substitution is aglutamine at position 44 of SEQ ID NO:1. In some embodiments, the aminoacid substitution is a glutamine at position 45 of SEQ ID NO:1. In someembodiments, the amino acid substitution is a glutamine at position 46of SEQ ID NO:1. In some embodiments, the amino acid substitution is aglutamine at position 47 of SEQ ID NO:1.

In some embodiments, the amino acid substitution is a glutamine at anyone of amino acid positions 42-47 of SEQ ID NO:99, 100, 294, 295, or296. In some embodiments, the amino acid substitution is a glutamine atany one of amino acid positions 42-47 of an amino acid sequence thatshares 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identity with SEQ ID NO:99, 100, 294,295, or 296. Thus, in some embodiments, the amino acid substitution is aglutamine at position 42 of SEQ ID NO:99, 100, 294, 295, or 296. In someembodiments, the amino acid substitution is a glutamine at position 43of SEQ ID NO:99, 100, 294, 295, or 296. In some embodiments, the aminoacid substitution is a glutamine at position 44 of SEQ ID NO:99, 100,294, 295, or 296. In some embodiments, the amino acid substitution is aglutamine at position 45 of SEQ ID NO:99, 100, 294, 295, or 296. In someembodiments, the amino acid substitution is a glutamine at position 46of SEQ ID NO:99, 100, 294, 295, or 296. In some embodiments, the aminoacid substitution is a glutamine at position 47 of SEQ ID NO:99, 100,294, 295, or 296.

In some embodiments, the amino acid substitution is a glutamate at anyone of amino acid positions 42-47 of SEQ ID NO:1. In some embodiments,the amino acid substitution is a glutamate at any one of amino acidpositions 42-47 of an amino acid sequence that shares 90-99%, e.g., atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identity with SEQ ID NO:1. Thus, in some embodiments, theamino acid substitution is a glutamate at position 42 of SEQ ID NO:1. Insome embodiments, the amino acid substitution is a glutamate at position43 of SEQ ID NO:1. In some embodiments, the amino acid substitution is aglutamate at position 44 of SEQ ID NO:1. In some embodiments, the aminoacid substitution is a glutamate at position 45 of SEQ ID NO:1. In someembodiments, the amino acid substitution is a glutamate at position 46of SEQ ID NO:1. In some embodiments, the amino acid substitution is aglutamate at position 47 of SEQ ID NO:1.

In some embodiments, the amino acid substitution is a glutamate at anyone of amino acid positions 42-47 of SEQ ID NO:99, 100, 294, 295, or296. In some embodiments, the amino acid substitution is a glutamate atany one of amino acid positions 42-47 of an amino acid sequence thatshares 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identity with SEQ ID NO:99, 100, 294,295, or 296. Thus, in some embodiments, the amino acid substitution is aglutamate at position 42 of SEQ ID NO:99, 100, 294, 295, or 296. In someembodiments, the amino acid substitution is a glutamate at position 43of SEQ ID NO:99, 100, 294, 295, or 296. In some embodiments, the aminoacid substitution is a glutamate at position 44 of SEQ ID NO:99, 100,294, 295, or 296. In some embodiments, the amino acid substitution is aglutamate at position 45 of SEQ ID NO:99, 100, 294, 295, or 296. In someembodiments, the amino acid substitution is a glutamate at position 46of SEQ ID NO:99, 100, 294, 295, or 296. In some embodiments, the aminoacid substitution is a glutamate at position 47 of SEQ ID NO:99, 100,294, 295, or 296.

In some embodiments, the amino acid substitution is a high-helixpropensity amino acid (e.g., alanine, isoleucine, leucine, arginine,methionine, lysine, glutamine, and/or glutamate) substitution at any oneof amino acid positions 257-262 (R257, A258, G259, A260, L261 and/orA262) of SEQ ID NO:1. In some embodiments, the amino acid substitutionis a high-helix propensity amino acid (e.g., alanine, isoleucine,leucine, arginine, methionine, lysine, glutamine, and/or glutamate)substitution at any one of amino acid positions 257-262 (R257, A258,G259, A260, L261 and/or A262) of an amino acid sequence that shares90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identity with SEQ ID NO:1. In someembodiments, the amino acid substitution is a high-helix propensityamino acid substitution at amino acid position 257 of SEQ ID NO:1. Insome embodiments, the amino acid substitution is a high-helix propensityamino acid substitution at amino acid position 258 of SEQ ID NO:1. Insome embodiments, the amino acid substitution is a high-helix propensityamino acid substitution at amino acid position 259 of SEQ ID NO:1. Insome embodiments, the amino acid substitution is a high-helix propensityamino acid substitution at amino acid position 260 of SEQ ID NO:1. Insome embodiments, the amino acid substitution is a high-helix propensityamino acid substitution at amino acid position 261 of SEQ ID NO:1. Insome embodiments, the amino acid substitution is a high-helix propensityamino acid substitution at amino acid position 262 of SEQ ID NO:1.

In some embodiments, the amino acid substitution is a high-helixpropensity amino acid (e.g., alanine, isoleucine, leucine, arginine,methionine, lysine, glutamine, and/or glutamate) substitution at any oneof amino acid positions 257-262 (R257, A258, G259, A260, L261 and/orA262) of SEQ ID NO:99, 100, 294, 295, or 296. In some embodiments, theamino acid substitution is a high-helix propensity amino acid (e.g.,alanine, isoleucine, leucine, arginine, methionine, lysine, glutamine,and/or glutamate) substitution at any one of amino acid positions257-262 (R257, A258, G259, A260, L261 and/or A262) of an amino acidsequence that shares 90-99%, e.g., at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identity with SEQ IDNO:99, 100, 294, 295, or 296. In some embodiments, the amino acidsubstitution is a high-helix propensity amino acid substitution at aminoacid position 257 of SEQ ID NO:99, 100, 294, 295, or 296. In someembodiments, the amino acid substitution is a high-helix propensityamino acid substitution at amino acid position 258 of SEQ ID NO:99, 100,294, 295, or 296. In some embodiments, the amino acid substitution is ahigh-helix propensity amino acid substitution at amino acid position 259of SEQ ID NO:99, 100, 294, 295, or 296. In some embodiments, the aminoacid substitution is a high-helix propensity amino acid substitution atamino acid position 260 of SEQ ID NO:99, 100, 294, 295, or 296. In someembodiments, the amino acid substitution is a high-helix propensityamino acid substitution at amino acid position 261 of SEQ ID NO:99, 100,294, 295, or 296. In some embodiments, the amino acid substitution is ahigh-helix propensity amino acid substitution at amino acid position 262of SEQ ID NO:99, 100, 294, 295, or 296.

In some embodiments, the amino acid substitution is an alanine at anyone of amino acid positions 257-262 of SEQ ID NO:1. In some embodiments,the amino acid substitution is an alanine at any one of amino acidpositions 257-262 of an amino acid sequence that shares 90-99%, e.g., atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identity with SEQ ID NO:1. Thus, in some embodiments, theamino acid substitution is an alanine at position 257 of SEQ ID NO:1. Insome embodiments, the amino acid substitution is an alanine at position258 of SEQ ID NO:1. In some embodiments, the amino acid substitution isan alanine at position 259 of SEQ ID NO:1. In some embodiments, theamino acid substitution is an alanine at position 260 of SEQ ID NO:1. Insome embodiments, the amino acid substitution is an alanine at position261 of SEQ ID NO:1. In some embodiments, the amino acid substitution isan alanine at position 262 of SEQ ID NO:1.

In some embodiments, the amino acid substitution is an alanine at anyone of amino acid positions 257-262 of SEQ ID NO:99, 100, 294, 295, or296. In some embodiments, the amino acid substitution is an alanine atany one of amino acid positions 257-262 of an amino acid sequence thatshares 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identity with SEQ ID NO:99, 100, 294,295, or 296. Thus, in some embodiments, the amino acid substitution isan alanine at position 257 of SEQ ID NO:99, 100, 294, 295, or 296. Insome embodiments, the amino acid substitution is an alanine at position258 of SEQ ID NO:99, 100, 294, 295, or 296. In some embodiments, theamino acid substitution is an alanine at position 259 of SEQ ID NO:99,100, 294, 295, or 296. In some embodiments, the amino acid substitutionis an alanine at position 260 of SEQ ID NO:99, 100, 294, 295, or 296. Insome embodiments, the amino acid substitution is an alanine at position261 of SEQ ID NO:99, 100, 294, 295, or 296. In some embodiments, theamino acid substitution is an alanine at position 262 of SEQ ID NO:99,100, 294, 295, or 296.

In some embodiments, the amino acid substitution is an isoleucine at anyone of amino acid positions 257-262 of SEQ ID NO:1. In some embodiments,the amino acid substitution is an isoleucine at any one of amino acidpositions 257-262 of an amino acid sequence that shares 90-99%, e.g., atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identity with SEQ ID NO:1. Thus, in some embodiments, theamino acid substitution is an isoleucine at position 257 of SEQ ID NO:1.In some embodiments, the amino acid substitution is an isoleucine atposition 258 of SEQ ID NO:1. In some embodiments, the amino acidsubstitution is an isoleucine at position 259 of SEQ ID NO:1. In someembodiments, the amino acid substitution is an isoleucine at position260 of SEQ ID NO:1. In some embodiments, the amino acid substitution isan isoleucine at position 261 of SEQ ID NO:1. In some embodiments, theamino acid substitution is an isoleucine at position 262 of SEQ ID NO:1.

In some embodiments, the amino acid substitution is an isoleucine at anyone of amino acid positions 257-262 of SEQ ID NO:99, 100, 294, 295, or296. In some embodiments, the amino acid substitution is an isoleucineat any one of amino acid positions 257-262 of an amino acid sequencethat shares 90-99%, e.g., at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% identity with SEQ ID NO:99,100, 294, 295, or 296. Thus, in some embodiments, the amino acidsubstitution is an isoleucine at position 257 of SEQ ID NO:99, 100, 294,295, or 296. In some embodiments, the amino acid substitution is anisoleucine at position 258 of SEQ ID NO:99, 100, 294, 295, or 296. Insome embodiments, the amino acid substitution is an isoleucine atposition 259 of SEQ ID NO:99, 100, 294, 295, or 296. In someembodiments, the amino acid substitution is an isoleucine at position260 of SEQ ID NO:99, 100, 294, 295, or 296. In some embodiments, theamino acid substitution is an isoleucine at position 261 of SEQ IDNO:99, 100, 294, 295, or 296. In some embodiments, the amino acidsubstitution is an isoleucine at position 262 of SEQ ID NO:99, 100, 294,295, or 296.

In some embodiments, the amino acid substitution is a leucine at any oneof amino acid positions 257-262 of SEQ ID NO:1. In some embodiments, theamino acid substitution is a leucine at any one of amino acid positions257-262 of an amino acid sequence that shares 90-99%, e.g., at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% identity with SEQ ID NO:1. Thus, in some embodiments, the amino acidsubstitution is a leucine at position 257 of SEQ ID NO:1. In someembodiments, the amino acid substitution is a leucine at position 258 ofSEQ ID NO:1. In some embodiments, the amino acid substitution is aleucine at position 259 of SEQ ID NO:1. In some embodiments, the aminoacid substitution is a leucine at position 260 of SEQ ID NO:1. In someembodiments, the amino acid substitution is a leucine at position 261 ofSEQ ID NO:1. In some embodiments, the amino acid substitution is aleucine at position 262 of SEQ ID NO:1.

In some embodiments, the amino acid substitution is a leucine at any oneof amino acid positions 257-262 of SEQ ID NO:99, 100, 294, 295, or 296.In some embodiments, the amino acid substitution is a leucine at any oneof amino acid positions 257-262 of an amino acid sequence that shares90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identity with SEQ ID NO:99, 100, 294, 295, or296. Thus, in some embodiments, the amino acid substitution is a leucineat position 257 of SEQ ID NO:99, 100, 294, 295, or 296. In someembodiments, the amino acid substitution is a leucine at position 258 ofSEQ ID NO:99, 100, 294, 295, or 296. In some embodiments, the amino acidsubstitution is a leucine at position 259 of SEQ ID NO:99, 100, 294,295, or 296. In some embodiments, the amino acid substitution is aleucine at position 260 of SEQ ID NO:99, 100, 294, 295, or 296. In someembodiments, the amino acid substitution is a leucine at position 261 ofSEQ ID NO:99, 100, 294, 295, or 296. In some embodiments, the amino acidsubstitution is a leucine at position 262 of SEQ ID NO:99, 100, 294,295, or 296.

In some embodiments, the amino acid substitution is an arginine at anyone of amino acid positions 257-262 of SEQ ID NO:1. In some embodiments,the amino acid substitution is a arginine at any one of amino acidpositions 257-262 of an amino acid sequence that shares 90-99%, e.g., atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identity with SEQ ID NO:1. Thus, in some embodiments, theamino acid substitution is an arginine at position 257 of SEQ ID NO:1.In some embodiments, the amino acid substitution is an arginine atposition 258 of SEQ ID NO:1. In some embodiments, the amino acidsubstitution is an arginine at position 259 of SEQ ID NO:1. In someembodiments, the amino acid substitution is an arginine at position 260of SEQ ID NO:1. In some embodiments, the amino acid substitution is anarginine at position 261 of SEQ ID NO:1. In some embodiments, the aminoacid substitution is an arginine at position 262 of SEQ ID NO:1.

In some embodiments, the amino acid substitution is an arginine at anyone of amino acid positions 257-262 of SEQ ID NO:99, 100, 294, 295, or296. In some embodiments, the amino acid substitution is an arginine atany one of amino acid positions 257-262 of an amino acid sequence thatshares 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identity with SEQ ID NO:99, 100, 294,295, or 296. Thus, in some embodiments, the amino acid substitution isan arginine at position 257 of SEQ ID NO:99, 100, 294, 295, or 296. Insome embodiments, the amino acid substitution is an arginine at position258 of SEQ ID NO:99, 100, 294, 295, or 296. In some embodiments, theamino acid substitution is an arginine at position 259 of SEQ ID NO:99,100, 294, 295, or 296. In some embodiments, the amino acid substitutionis an arginine at position 260 of SEQ ID NO:99, 100, 294, 295, or 296.In some embodiments, the amino acid substitution is an arginine atposition 261 of SEQ ID NO:99, 100, 294, 295, or 296. In someembodiments, the amino acid substitution is an arginine at position 262of SEQ ID NO:99, 100, 294, 295, or 296.

In some embodiments, the amino acid substitution is a methionine at anyone of amino acid positions 257-262 of SEQ ID NO:1. In some embodiments,the amino acid substitution is a methionine at any one of amino acidpositions 257-262 of an amino acid sequence that shares 90-99%, e.g., atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identity with SEQ ID NO:1. Thus, in some embodiments, theamino acid substitution is a methionine at position 257 of SEQ ID NO:1.In some embodiments, the amino acid substitution is a methionine atposition 258 of SEQ ID NO:1. In some embodiments, the amino acidsubstitution is a methionine at position 259 of SEQ ID NO:1. In someembodiments, the amino acid substitution is a methionine at position 260of SEQ ID NO:1. In some embodiments, the amino acid substitution is amethionine at position 261 of SEQ ID NO:1. In some embodiments, theamino acid substitution is a methionine at position 262 of SEQ ID NO:1.

In some embodiments, the amino acid substitution is a methionine at anyone of amino acid positions 257-262 of SEQ ID NO:99, 100, 294, 295, or296. In some embodiments, the amino acid substitution is a methionine atany one of amino acid positions 257-262 of an amino acid sequence thatshares 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identity with SEQ ID NO:99, 100, 294,295, or 296. Thus, in some embodiments, the amino acid substitution is amethionine at position 257 of SEQ ID NO:99, 100, 294, 295, or 296. Insome embodiments, the amino acid substitution is a methionine atposition 258 of SEQ ID NO:99, 100, 294, 295, or 296. In someembodiments, the amino acid substitution is a methionine at position 259of SEQ ID NO:99, 100, 294, 295, or 296. In some embodiments, the aminoacid substitution is a methionine at position 260 of SEQ ID NO:99, 100,294, 295, or 296. In some embodiments, the amino acid substitution is amethionine at position 261 of SEQ ID NO:99, 100, 294, 295, or 296. Insome embodiments, the amino acid substitution is a methionine atposition 262 of SEQ ID NO:99, 100, 294, 295, or 296.

In some embodiments, the amino acid substitution is a lysine at any oneof amino acid positions 257-262 of SEQ ID NO:1. In some embodiments, theamino acid substitution is a lysine at any one of amino acid positions257-262 of an amino acid sequence that shares 90-99%, e.g., at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% identity with SEQ ID NO:1. Thus, in some embodiments, the amino acidsubstitution is a lysine at position 257 of SEQ ID NO:1. In someembodiments, the amino acid substitution is a lysine at position 258 ofSEQ ID NO:1. In some embodiments, the amino acid substitution is alysine at position 259 of SEQ ID NO:1. In some embodiments, the aminoacid substitution is a lysine at position 260 of SEQ ID NO:1. In someembodiments, the amino acid substitution is a lysine at position 261 ofSEQ ID NO:1. In some embodiments, the amino acid substitution is alysine at position 262 of SEQ ID NO:1.

In some embodiments, the amino acid substitution is a lysine at any oneof amino acid positions 257-262 of SEQ ID NO:99, 100, 294, 295, or 296.In some embodiments, the amino acid substitution is a lysine at any oneof amino acid positions 257-262 of an amino acid sequence that shares90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identity with SEQ ID NO:99, 100, 294, 295, or296. Thus, in some embodiments, the amino acid substitution is a lysineat position 257 of SEQ ID NO:99, 100, 294, 295, or 296. In someembodiments, the amino acid substitution is a lysine at position 258 ofSEQ ID NO:99, 100, 294, 295, or 296. In some embodiments, the amino acidsubstitution is a lysine at position 259 of SEQ ID NO:99, 100, 294, 295,or 296. In some embodiments, the amino acid substitution is a lysine atposition 260 of SEQ ID NO:99, 100, 294, 295, or 296. In someembodiments, the amino acid substitution is a lysine at position 261 ofSEQ ID NO:99, 100, 294, 295, or 296. In some embodiments, the amino acidsubstitution is a lysine at position 262 of SEQ ID NO:99, 100, 294, 295,or 296.

In some embodiments, the amino acid substitution is a glutamine at anyone of amino acid positions 257-262 of SEQ ID NO:1. In some embodiments,the amino acid substitution is a glutamine at any one of amino acidpositions 257-262 of an amino acid sequence that shares 90-99%, e.g., atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identity with SEQ ID NO:1. Thus, in some embodiments, theamino acid substitution is a glutamine at position 257 of SEQ ID NO:1.In some embodiments, the amino acid substitution is a glutamine atposition 258 of SEQ ID NO:1. In some embodiments, the amino acidsubstitution is a glutamine at position 259 of SEQ ID NO:1. In someembodiments, the amino acid substitution is a glutamine at position 260of SEQ ID NO:1. In some embodiments, the amino acid substitution is aglutamine at position 261 of SEQ ID NO:1. In some embodiments, the aminoacid substitution is a glutamine at position 262 of SEQ ID NO:1.

In some embodiments, the amino acid substitution is a glutamine at anyone of amino acid positions 257-262 of SEQ ID NO:99, 100, 294, 295, or296. In some embodiments, the amino acid substitution is a glutamine atany one of amino acid positions 257-262 of an amino acid sequence thatshares 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identity with SEQ ID NO:99, 100, 294,295, or 296. Thus, in some embodiments, the amino acid substitution is aglutamine at position 257 of SEQ ID NO:99, 100, 294, 295, or 296. Insome embodiments, the amino acid substitution is a glutamine at position258 of SEQ ID NO:99, 100, 294, 295, or 296. In some embodiments, theamino acid substitution is a glutamine at position 259 of SEQ ID NO:99,100, 294, 295, or 296. In some embodiments, the amino acid substitutionis a glutamine at position 260 of SEQ ID NO:99, 100, 294, 295, or 296.In some embodiments, the amino acid substitution is a glutamine atposition 261 of SEQ ID NO:99, 100, 294, 295, or 296. In someembodiments, the amino acid substitution is a glutamine at position 262of SEQ ID NO:99, 100, 294, 295, or 296.

In some embodiments, the amino acid substitution is a glutamate at anyone of amino acid positions 257-262 of SEQ ID NO:1. In some embodiments,the amino acid substitution is a glutamate at any one of amino acidpositions 257-262 of an amino acid sequence that shares 90-99%, e.g., atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identity with SEQ ID NO:1. Thus, in some embodiments, theamino acid substitution is a glutamate at position 257 of SEQ ID NO:1.In some embodiments, the amino acid substitution is a glutamate atposition 258 of SEQ ID NO:1. In some embodiments, the amino acidsubstitution is a glutamate at position 259 of SEQ ID NO:1. In someembodiments, the amino acid substitution is a glutamate at position 260of SEQ ID NO:1. In some embodiments, the amino acid substitution is aglutamate at position 261 of SEQ ID NO:1. In some embodiments, the aminoacid substitution is a glutamate at position 262 of SEQ ID NO:1.

In some embodiments, the amino acid substitution is a glutamate at anyone of amino acid positions 257-262 of SEQ ID NO:99, 100, 294, 295, or296. In some embodiments, the amino acid substitution is a glutamate atany one of amino acid positions 257-262 of an amino acid sequence thatshares 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identity with SEQ ID NO:99, 100, 294,295, or 296. Thus, in some embodiments, the amino acid substitution is aglutamate at position 257 of SEQ ID NO:99, 100, 294, 295, or 296. Insome embodiments, the amino acid substitution is a glutamate at position258 of SEQ ID NO:99, 100, 294, 295, or 296. In some embodiments, theamino acid substitution is a glutamate at position 259 of SEQ ID NO:99,100, 294, 295, or 296. In some embodiments, the amino acid substitutionis a glutamate at position 260 of SEQ ID NO:99, 100, 294, 295, or 296.In some embodiments, the amino acid substitution is a glutamate atposition 261 of SEQ ID NO:99, 100, 294, 295, or 296. In someembodiments, the amino acid substitution is a glutamate at position 262of SEQ ID NO:99, 100, 294, 295, or 296.

In some embodiments, the T7 RNA polymerase variant comprises an aminoacid sequence of SEQ ID NO:1 modified to include an amino acidsubstitution of a high-helix propensity amino acid at position G47, S43,R257, and/or G259. In some embodiments, the T7 RNA polymerase variantcomprises an amino acid sequence that shares 90-99%, e.g., at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with SEQ ID NO:1 modified to include an amino acid substitutionof a high-helix propensity amino acid at position G47, S43, R257, and/orG259.

In some embodiments, the T7 RNA polymerase variant comprises an aminoacid sequence of SEQ ID NO:99, 100, 294, 295, or 296 modified to includean amino acid substitution of a high-helix propensity amino acid atposition G47, S43, R257, and/or G259. In some embodiments, the T7 RNApolymerase variant comprises an amino acid sequence that shares 90-99%,e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% identity with SEQ ID NO:99, 100, 294, 295, or 296modified to include an amino acid substitution of a high-helixpropensity amino acid at position G47, S43, R257, and/or G259.

In some embodiments, a T7 RNA polymerase variant comprises an amino acidsequence of SEQ ID NO:2. In some embodiments, a T7 RNA polymerasevariant comprises an amino acid sequence of SEQ ID NO:3. In someembodiments, a T7 RNA polymerase variant comprises an amino acidsequence of SEQ ID NO:4. In some embodiments, a T7 RNA polymerasevariant comprises an amino acid sequence of SEQ ID NO:5.

In some embodiments, a T7 RNA polymerase variant comprises an amino acidsequence of SEQ ID NO:107 or 108. In some embodiments, a T7 RNApolymerase variant comprises an amino acid sequence of SEQ ID NO:109 or110. In some embodiments, a T7 RNA polymerase variant comprises an aminoacid sequence of SEQ ID NO:111 or 112. In some embodiments, a T7 RNApolymerase variant comprises an amino acid sequence of SEQ ID NO:113 or114.

Also provided herein are RNA polymerase variants with at least 2 (2 ormore) substitutions. In some embodiments, a RNA polymerase comprise atleast 2 high-helix propensity amino acid (e.g., alanine, isoleucine,leucine, arginine, methionine, lysine, glutamine, and/or glutamate)substitutions at any one of amino acid positions 42-47 (E42, S43, Y44,E45, M46 and/or G47) and/or amino acid positions 257-262 (R257, A258,G259, A260, L261 and/or A262) of SEQ ID NO:1 or an amino acid sequencethat shares 90-99%, e.g., at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% identity with SEQ ID NO:1.

For example, a RNA polymerase variant may comprise an amino acidsubstitution (e.g., alanine, isoleucine, leucine, arginine, methionine,lysine, glutamine, and/or glutamate) at positions E42 and S43, E42 andY44, E42 and E45, E42 and M46, E42 and G47, S43 and Y44, S43 and E45,S43 and M46, S43 and G47, Y44 and E45, Y44 and M46, Y44 and G47, E45 andM46, E45 and G47, or M46 and G47 of SEQ ID NO:1 or an amino acidsequence that shares 90-99%, e.g., at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identity with SEQ IDNO:1.

In some embodiments, a RNA polymerase variant comprises an amino acidsubstitution (e.g., alanine, isoleucine, leucine, arginine, methionine,lysine, glutamine, and/or glutamate) at positions R257 and A258, R257and G259, R257 and A260, R257 and L261, R257 and A262, A258 and G259,A258 and A260, A258 and L261, A258 and A262, G259 and A260, G259 andL261, G259 and A262, A260 and L261, A260 and A262, or L261 and A262 ofSEQ ID NO:1 or an amino acid sequence that shares 90-99%, e.g., at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% identity with SEQ ID NO:1.

In some embodiments, a RNA polymerase variant comprises an amino acidsubstitution (e.g., alanine, isoleucine, leucine, arginine, methionine,lysine, glutamine, and/or glutamate) at positions E42 and R257, E42 andA258, E42 and G259, E42 and A260, E42 and L261, E42 and A262, S43 andR257, S43 and A258, S43 and G259, S43 and A260, S43 and L261, S43 andA262, Y44 and R257, Y44 and A258, Y44 and G259, Y44 and A260, Y44 andL261, Y44 and A262, E45 and R257, E45 and A258, E45 and G259, E45 andA260, E45 and L261, E45 and A262, M46 and R257, M46 and A258, M46 andG259, M46 and A260, M46 and L261, M46 and A262, G47 and R257, G47 andA258, G47 and G259, G47 and A260, G47 and L261, or G47 and A262 of SEQID NO:1 or an amino acid sequence that shares 90-99%, e.g., at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% identity with SEQ ID NO:1.

In some embodiments, a RNA polymerase comprise amino acid substitutionsS43A and G47A of SEQ ID NO:1 or an amino acid sequence that shares90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identity with SEQ ID NO:1. In someembodiments, a RNA polymerase comprise amino acid substitutions S43A andR257A of SEQ ID NO:1 or an amino acid sequence that shares 90-99%, e.g.,at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99% identity with SEQ ID NO:1. In some embodiments, a RNApolymerase comprise amino acid substitutions S43A and G259A of SEQ IDNO:1 or an amino acid sequence that shares 90-99%, e.g., at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with SEQ ID NO:1. In some embodiments, a RNA polymerasecomprise amino acid substitutions G47A and R257A of SEQ ID NO:1 or anamino acid sequence that shares 90-99%, e.g., at least 90%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% identitywith SEQ ID NO:1. In some embodiments, a RNA polymerase comprise aminoacid substitutions G47A and R257A of SEQ ID NO:1 or an amino acidsequence that shares 90-99%, e.g., at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identity with SEQ IDNO:1. In some embodiments, a RNA polymerase comprise amino acidsubstitutions R257A and G259A of SEQ ID NO:1 or an amino acid sequencethat shares 90-99%, e.g., at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% identity with SEQ ID NO:1. Insome embodiments, a RNA polymerase comprise amino acid substitutionsG47A and G259A of SEQ ID NO:1 or an amino acid sequence that shares90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identity with SEQ ID NO:1.

In some embodiments, a RNA polymerase variant comprises at least 3 (orat least 4 or at least 5) high-helix propensity amino acid (e.g.,alanine, isoleucine, leucine, arginine, methionine, lysine, glutamine,and/or glutamate) substitutions at any one of amino acid positions 42-47(E42, S43, Y44, E45, M46 and/or G47) and/or amino acid positions 257-262(R257, A258, G259, A260, L261 and/or A262) of SEQ ID NO:1 or an aminoacid sequence that shares 90-99%, e.g., at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identity with SEQID NO:1.

In some embodiments, a RNA polymerase variant comprises at least 1 (orat least 2 or at least 3 or at least 4 or at least 5) amino acidsubstitution(s) in SEQ ID NO:1 or an amino acid sequence that shares90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identity with SEQ ID NO:1, selected from thefollowing: E42R, S43A, S43E, S43L, S43R, E45R, E45L, M46A, G47A, G47E,G47L, G47R, N165W, E167M, E167N, E168I, E168T, E168V, A181F, A181W,G184M, E187F, A255Q, A255K, A255I, A255Y, R257A, R257E, R257L, R257W,G259A, G259E, G259L, G259R, A260W, and A260R.

In some embodiments, a RNA polymerase comprise at least 2 high-helixpropensity amino acid (e.g., alanine, isoleucine, leucine, arginine,methionine, lysine, glutamine, and/or glutamate) substitutions at anyone of amino acid positions 42-47 (E42, S43, Y44, E45, M46 and/or G47)and/or amino acid positions 257-262 (R257, A258, G259, A260, L261 and/orA262) of SEQ ID NO:99, 100, 294, 295, or 296 or an amino acid sequencethat shares 90-99%, e.g., at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% identity with SEQ ID NO:99,100, 294, 295, or 296.

For example, a RNA polymerase variant may comprise an amino acidsubstitution (e.g., alanine, isoleucine, leucine, arginine, methionine,lysine, glutamine, and/or glutamate) at positions E42 and S43, E42 andY44, E42 and E45, E42 and M46, E42 and G47, S43 and Y44, S43 and E45,S43 and M46, S43 and G47, Y44 and E45, Y44 and M46, Y44 and G47, E45 andM46, E45 and G47, or M46 and G47 of SEQ ID NO:99, 100, 294, 295, or 296or an amino acid sequence that shares 90-99%, e.g., at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity with SEQ ID NO:99, 100, 294, 295, or 296.

In some embodiments, a RNA polymerase variant comprises an amino acidsubstitution (e.g., alanine, isoleucine, leucine, arginine, methionine,lysine, glutamine, and/or glutamate) at positions R257 and A258, R257and G259, R257 and A260, R257 and L261, R257 and A262, A258 and G259,A258 and A260, A258 and L261, A258 and A262, G259 and A260, G259 andL261, G259 and A262, A260 and L261, A260 and A262, or L261 and A262 ofSEQ ID NO:99, 100, 294, 295, or 296 or an amino acid sequence thatshares 90-99%, e.g., at least 90%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identity with SEQ ID NO:99, 100, 294,295, or 296.

In some embodiments, a RNA polymerase variant comprises an amino acidsubstitution (e.g., alanine, isoleucine, leucine, arginine, methionine,lysine, glutamine, and/or glutamate) at positions E42 and R257, E42 andA258, E42 and G259, E42 and A260, E42 and L261, E42 and A262, S43 andR257, S43 and A258, S43 and G259, S43 and A260, S43 and L261, S43 andA262, Y44 and R257, Y44 and A258, Y44 and G259, Y44 and A260, Y44 andL261, Y44 and A262, E45 and R257, E45 and A258, E45 and G259, E45 andA260, E45 and L261, E45 and A262, M46 and R257, M46 and A258, M46 andG259, M46 and A260, M46 and L261, M46 and A262, G47 and R257, G47 andA258, G47 and G259, G47 and A260, G47 and L261, or G47 and A262 of SEQID NO:99, 100, 294, 295, or 296 or an amino acid sequence that shares90-99%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identity with SEQ ID NO:99, 100, 294, 295, or296.

In some embodiments, a RNA polymerase comprises amino acid substitutionsS43A and G47A of SEQ ID NO:99, 100, 294, 295, or 296 or an amino acidsequence that shares 90-99%, e.g., at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identity with SEQ IDNO:99, 100, 294, 295, or 296. In some embodiments, a RNA polymerasecomprises amino acid substitutions S43A and R257A of SEQ ID NO:99, 100,294, 295, or 296 or an amino acid sequence that shares 90-99%, e.g., atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identity with SEQ ID NO:99, 100, 294, 295, or 296. In someembodiments, a RNA polymerase comprises amino acid substitutions S43Aand G259A of SEQ ID NO:99, 100, 294, 295, or 296 or an amino acidsequence that shares 90-99%, e.g., at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identity with SEQ IDNO:99, 100, 294, 295, or 296. In some embodiments, a RNA polymerasecomprises amino acid substitutions G47A and R257A of SEQ ID NO:99, 100,294, 295, or 296 or an amino acid sequence that shares 90-99%, e.g., atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identity with SEQ ID NO:99, 100, 294, 295, or 296. In someembodiments, a RNA polymerase comprises amino acid substitutions G47Aand R257A of SEQ ID NO:99, 100, 294, 295, or 296 or an amino acidsequence that shares 90-99%, e.g., at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identity with SEQ IDNO:99, 100, 294, 295, or 296. In some embodiments, a RNA polymerasecomprises amino acid substitutions R257A and G259A of SEQ ID NO:99, 100,294, 295, or 296 or an amino acid sequence that shares 90-99%, e.g., atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identity with SEQ ID NO:99, 100, 294, 295, or 296. In someembodiments, a RNA polymerase comprises amino acid substitutions G47Aand G259A of SEQ ID NO:99, 100, 294, 295, or 296 or an amino acidsequence that shares 90-99%, e.g., at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identity with SEQ IDNO:99, 100, 294, 295, or 296.

In some embodiments, a RNA polymerase variant comprises at least 3 (orat least 4 or at least 5) high-helix propensity amino acid (e.g.,alanine, isoleucine, leucine, arginine, methionine, lysine, glutamine,and/or glutamate) substitutions at any one of amino acid positions 42-47(E42, S43, Y44, E45, M46 and/or G47) and/or amino acid positions 257-262(R257, A258, G259, A260, L261 and/or A262) of SEQ ID NO:99, 100, 294,295, or 296 or an amino acid sequence that shares 90-99%, e.g., at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% identity with SEQ ID NO:99, 100, 294, 295, or 296.

In some embodiments, a RNA polymerase variant comprises at least 1 (orat least 2 or at least 3 or at least 4 or at least 5) amino acidsubstitution(s) in SEQ ID NO:99, 100, 294, 295, or 296 or an amino acidsequence that shares 90-99%, e.g., at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identity with SEQ IDNO:99, 100, 294, 295, or 296, selected from the following: E42R, S43A,S43E, S43L, S43R, E45R, E45L, M46A, G47A, G47E, G47L, G47R, N165W,E167M, E167N, E1681, E168T, E168V, A181F, A181W, G184M, E187F, R257A,R257E, R257L, R257W, G259A, G259E, G259L, G259R, A260W, and A260R.

In some embodiments, a RNA polymerase variant comprises at least 1 (orat least 2 or at least 3 or at least 4 or at least 5) amino acidsubstitution(s) selected from the following: E42R, S43A, S43E, S43L,S43R, E45R, E45L, M46A, G47A, G47E, G47L, G47R, N165W, E167M, E167N,E168I, E168T, E168V, A181F, A181W, G184M, E187F, R257A, R257E, R257L,R257W, G259A, G259E, G259L, G259R, A260W, and A260R, further comprisesat least 1 (or at least 2 or at least 3 or at least 4 or at least 5)other amino acid substitution (e.g., an amino acid substitution notprovided herein).

Thus, the present disclosure encompasses RNA polymerase variants thatcomprise at least 1 (or at least 2 or at least 3 or at least 4 or atleast 5) amino acid substitution(s) selected from the following: E42R,S43A, S43E, S43L, S43R, E45R, E45L, M46A, G47A, G47E, G47L, G47R, N165W,E167M, E167N, E168I, E168T, E168V, A181F, A181W, G184M, E187F, A255K,A255Q, A255Y, A255I, R257A, R257E, R257L, R257W, G259A, G259E, G259L,G259R, A260W, and A260R and are at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% identical to the amino acid sequence of SEQ ID NO: 1.

The term “identity” refers to a relationship between the sequences oftwo or more polypeptides (e.g. enzymes) or polynucleotides (nucleicacids), as determined by comparing the sequences. Identity also refersto the degree of sequence relatedness between or among sequences asdetermined by the number of matches between strings of two or more aminoacid residues or nucleic acid residues. Identity measures the percent ofidentical matches between the smaller of two or more sequences with gapalignments (if any) addressed by a particular mathematical model orcomputer program (e.g., “algorithms”). Identity of related proteins ornucleic acids can be readily calculated by known methods. “Percent (%)identity” as it applies to polypeptide or polynucleotide sequences isdefined as the percentage of residues (amino acid residues or nucleicacid residues) in the candidate amino acid or nucleic acid sequence thatare identical with the residues in the amino acid sequence or nucleicacid sequence of a second sequence after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent identity.Methods and computer programs for the alignment are well known in theart. It is understood that identity depends on a calculation of percentidentity but may differ in value due to gaps and penalties introduced inthe calculation. Generally, variants of a particular polynucleotide orpolypeptide (e.g., antigen) have at least 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% butless than 100% sequence identity to that particular referencepolynucleotide or polypeptide as determined by sequence alignmentprograms and parameters described herein and known to those skilled inthe art. Such tools for alignment include those of the BLAST suite(Stephen F. Altschul, et al (1997), “Gapped BLAST and PSI-BLAST: a newgeneration of protein database search programs”, Nucleic Acids Res.25:3389-3402). Another popular local alignment technique is based on theSmith-Waterman algorithm (Smith, T. F. & Waterman, M. S. (1981)“Identification of common molecular subsequences.” J. Mol. Biol.147:195-197). A general global alignment technique based on dynamicprogramming is the Needleman-Wunsch algorithm (Needleman, S. B. &Wunsch, C. D. (1970) “A general method applicable to the search forsimilarities in the amino acid sequences of two proteins.” J. Mol. Biol.48:443-453). More recently a Fast Optimal Global Sequence AlignmentAlgorithm (FOGSAA) has been developed that purportedly produces globalalignment of nucleotide and protein sequences faster than other optimalglobal alignment methods, including the Needleman-Wunsch algorithm.

Trinucleotide Caps

Also provided herein are co-transcriptional capping methods forribonucleic acid (RNA) synthesis. That is, RNA is produced in a“one-pot” reaction, without the need for a separate capping reaction.Thus, the methods, in some embodiments, comprise reacting apolynucleotide template with a T7 RNA polymerase variant, nucleosidetriphosphates, and a cap analog under in vitro transcription reactionconditions to produce RNA transcript.

A cap analog may be, for example, a dinucleotide cap, a trinucleotidecap, or a tetranucleotide cap. In some embodiments, a cap analog is adinucleotide cap. In some embodiments, a cap analog is a trinucleotidecap. In some embodiments, a cap analog is a tetranucleotide cap.

A trinucleotide cap, in some embodiments, comprises a compound offormula (I)

or a stereoisomer, tautomer or salt thereof, wherein

ring B₁ is a modified or unmodified Guanine;

ring B₂ and ring B₃ each independently is a nucleobase or a modifiednucleobase;

X₂ is O, S(O)_(p), NR₂₄ or CR₂₅R₂₆ in which p is 0, 1, or 2;

Y₀ is O or CR₆R₇;

Y1 is O, S(O)_(n), CR₆R₇, or NR₈, in which n is 0, 1, or 2;

each

is a single bond or absent, wherein when each

is a single bond, Y₁ is O, S(O)_(n), CR₆R₇, or NR₈; and when each

is absent, Y₁ is void;

Y₂ is (OP(O)R₄)_(m) in which m is 0, 1, or 2, or—O—(CR₄₀R₄₁)u-Q₀-(CR₄₂R₄₃)v-, in which Q₀ is a bond, O, S(O)_(r), NR₄₄,or CR₄₅R₄₆, r is 0, 1, or 2, and each of u and v independently is 1, 2,3 or 4;

each R₂ and R₂′ independently is halo, LNA, or OR₃;

each R₃ independently is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyland R₃, when being C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl, isoptionally substituted with one or more of halo, OH and C₁-C₆ alkoxylthat is optionally substituted with one or more OH or OC(O)—C₁-C₆ alkyl;

each R₄ and R₄′ independently is H, halo, C₁-C₆ alkyl, OH, SH, SeH, orBH₃ ⁻;

each of R₆, R₇, and R₈, independently, is -Q₁-T₁, in which Q₁ is a bondor C₁-C₃ alkyl linker optionally substituted with one or more of halo,cyano, OH and C₁-C₆ alkoxy, and T₁ is H, halo, OH, COOH, cyano, orR_(s1), in which R_(s1) is C₁-C₃ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,C₁-C₆ alkoxyl, C(O)O—C₁-C₆ alkyl, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl,NR₃₁R₃₂, (NR₃₁R₃₂R₃₃)⁺, 4 to 12-membered heterocycloalkyl, or 5- or6-membered heteroaryl, and R_(s1) is optionally substituted with one ormore substituents selected from the group consisting of halo, OH, oxo,C₁-C₆ alkyl, COOH, C(O)O—C₁-C₆ alkyl, cyano, C₁-C₆ alkoxyl, NR₃₁R₃₂,(NR₃₁R₃₂R₃₃)⁺, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 12-memberedheterocycloalkyl, and 5- or 6-membered heteroaryl;

each of R₁₀, R₁₁, R₁₂, R₁₃ R₁₄, and R₁₅, independently, is -Q₂-T₂, inwhich Q₂ is a bond or C₁-C₃ alkyl linker optionally substituted with oneor more of halo, cyano, OH and C₁-C₆ alkoxy, and T₂ is H, halo, OH, NH₂,cyano, NO₂, N₃, R_(s2), or OR_(s2), in which R_(s2) is C₁-C₆ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl,NHC(O)—C₁-C₆ alkyl, NR₃₁R₃₂, (NR₃₁R₃₂R₃₃)⁺, 4 to 12-memberedheterocycloalkyl, or 5- or 6-membered heteroaryl, and R_(s2) isoptionally substituted with one or more substituents selected from thegroup consisting of halo, OH, oxo, C₁-C₆ alkyl, COOH, C(O)O—C₁-C₆ alkyl,cyano, C₁-C₆ alkoxyl, NR₃₁R₃₂, (NR₃₁R₃₂R₃₃)⁺, C₃-C₈ cycloalkyl, C₆-C₁₀aryl, 4 to 12-membered heterocycloalkyl, and 5- or 6-memberedheteroaryl; or alternatively R₁₂ together with R₁₄ is oxo, or R₁₃together with R₁₅ is oxo,

each of R₂₀, R₂₁, R₂₂, and R₂₃ independently is -Q₃-T₃, in which Q₃ is abond or C₁-C₃ alkyl linker optionally substituted with one or more ofhalo, cyano, OH and C₁-C₆ alkoxy, and T₃ is H, halo, OH, NH₂, cyano,NO₂, N₃, R_(S3), or OR_(S3), in which R_(S3) is C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, NHC(O)—C₁-C₆alkyl, mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino, 4 to 12-memberedheterocycloalkyl, or 5- or 6-membered heteroaryl, and R_(S3) isoptionally substituted with one or more substituents selected from thegroup consisting of halo, OH, oxo, C₁-C₆ alkyl, COOH, C(O)O—C₁-C₆ alkyl,cyano, C₁-C₆ alkoxyl, amino, mono-C₁-C₆ alkylamino, di-C₁-C₆ alkylamino,C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to 12-membered heterocycloalkyl, and 5-or 6-membered heteroaryl; each of R₂₄, R₂₅, and R₂₆ independently is Hor C₁-C₆ alkyl;

each of R₂₇ and R₂₈ independently is H or OR₂₉; or R₂₇ and R₂₈ togetherform O—R₃₀—O; each R₂₉ independently is H, C₁-C₆ alkyl, C₂-C₆ alkenyl,or C₂-C₆ alkynyl and R₂₉, when being C₁-C₆ alkyl, C₂-C₆ alkenyl, orC₂-C₆ alkynyl, is optionally substituted with one or more of halo, OHand C₁-C₆ alkoxyl that is optionally substituted with one or more OH orOC(O)—C₁-C₆ alkyl;

R₃₀ is C₁-C₆ alkylene optionally substituted with one or more of halo,OH and C₁-C₆ alkoxyl;

each of R₃₁, R₃₂, and R₃₃, independently is H, C₁-C₆ alkyl, C₃-C₈cycloalkyl, C₆-C₁₀ aryl, 4 to 12-membered heterocycloalkyl, or 5- or6-membered heteroaryl;

each of R₄₀, R₄₁, R₄₂, and R₄₃ independently is H, halo, OH, cyano, N3,OP(O)R₄₇R₄₈, or C₁-C₆ alkyl optionally substituted with one or moreOP(O)R₄₇R₄₈, or one R₄₁ and one R₄₃, together with the carbon atoms towhich they are attached and Q₀, form C₄-C₁₀ cycloalkyl, 4- to14-membered heterocycloalkyl, C₆-C₁₀ aryl, or 5- to 14-memberedheteroaryl, and each of the cycloalkyl, heterocycloalkyl, phenyl, or 5-to 6-membered heteroaryl is optionally substituted with one or more ofOH, halo, cyano, N₃, oxo, OP(O)R₄₇R₄₈, C₁-C₆ alkyl, C₁-C₆ haloalkyl,COOH, C(O)O—C₁-C₆ alkyl, C₁-C₆ alkoxyl, C₁-C₆ haloalkoxyl, amino,mono-C₁-C₆ alkylamino, and di-C₁-C₆ alkylamino;

R₄₄ is H, C₁-C₆ alkyl, or an amine protecting group;

each of R₄₅ and R₄₆ independently is H, OP(O)R₄₇R₄₈, or C₁-C₆ alkyloptionally substituted with one or more OP(O)R₄₇R₄₈, and

each of R₄₇ and R₄₈, independently is H, halo, C₁-C₆ alkyl, OH, SH, SeH,or BH₃ ⁻.

It should be understood that a cap analog, as provided herein, mayinclude any of the cap analogs described in international publication WO2017/066797, published on 20 Apr. 2017, incorporated by reference hereinin its entirety.

In some embodiments, the B₂ middle position can be a non-ribosemolecule, such as arabinose.

In some embodiments R₂ is ethyl-based.

Thus, in some embodiments, a trinucleotide cap comprises the followingstructure:

In other embodiments, a trinucleotide cap comprises the followingstructure:

In yet other embodiments, a trinucleotide cap comprises the followingstructure:

In still other embodiments, a trinucleotide cap comprises the followingstructure:

A trinucleotide cap, in some embodiments, comprises a sequence selectedfrom the following sequences: GAA, GAC, GAG, GAU, GCA, GCC, GCG, GCU,GGA, GGC, GGG, GGU, GUA, GUC, GUG, and GUU. In some embodiments, atrinucleotide cap comprises GAA. In some embodiments, a trinucleotidecap comprises GAC. In some embodiments, a trinucleotide cap comprisesGAG. In some embodiments, a trinucleotide cap comprises GAU. In someembodiments, a trinucleotide cap comprises GCA. In some embodiments, atrinucleotide cap comprises GCC. In some embodiments, a trinucleotidecap comprises GCG. In some embodiments, a trinucleotide cap comprisesGCU. In some embodiments, a trinucleotide cap comprises GGA. In someembodiments, a trinucleotide cap comprises GGC. In some embodiments, atrinucleotide cap comprises GGG. In some embodiments, a trinucleotidecap comprises GGU. In some embodiments, a trinucleotide cap comprisesGUA. In some embodiments, a trinucleotide cap comprises GUC. In someembodiments, a trinucleotide cap comprises GUG. In some embodiments, atrinucleotide cap comprises GUU.

In some embodiments, a trinucleotide cap comprises a sequence selectedfrom the following sequences: m⁷GpppApA, m⁷GpppApC, m⁷GpppApG,m⁷GpppApU, m⁷GpppCpA, m⁷GpppCpC, m⁷GpppCpG, m⁷GpppCpU, m⁷GpppGpA,m⁷GpppGpC, m⁷GpppGpG, m⁷GpppGpU, m⁷GpppUpA, m⁷GpppUpC, m⁷GpppUpG, andm⁷GpppUpU.

In some embodiments, a trinucleotide cap comprises m⁷GpppApA. In someembodiments, a trinucleotide cap comprises m⁷GpppApC. In someembodiments, a trinucleotide cap comprises m⁷GpppApG. In someembodiments, a trinucleotide cap comprises m⁷GpppApU. In someembodiments, a trinucleotide cap comprises m⁷GpppCpA. In someembodiments, a trinucleotide cap comprises m⁷GpppCpC. In someembodiments, a trinucleotide cap comprises m⁷GpppCpG. In someembodiments, a trinucleotide cap comprises m⁷GpppCpU. In someembodiments, a trinucleotide cap comprises m⁷GpppGpA. In someembodiments, a trinucleotide cap comprises m⁷GpppGpC. In someembodiments, a trinucleotide cap comprises m⁷GpppGpG. In someembodiments, a trinucleotide cap comprises m⁷GpppGpU. In someembodiments, a trinucleotide cap comprises m⁷GpppUpA. In someembodiments, a trinucleotide cap comprises m⁷GpppUpC. In someembodiments, a trinucleotide cap comprises m⁷GpppUpG. In someembodiments, a trinucleotide cap comprises m⁷GpppUpU.

A trinucleotide cap, in some embodiments, comprises a sequence selectedfrom the following sequences: m⁷G_(3′OMe)pppApA, m⁷G_(3′OMe)pppApC,m⁷G_(3′OMe)pppApG, m⁷G_(3′OMe)pppApU, m⁷G_(3′OMe)pppCpA,m⁷G_(3′OMe)pppCpC, m⁷G_(3′OMe)pppCpG, m⁷G_(3′OMe)pppCpU,m⁷G_(3′OMe)pppGpA, m⁷G_(3′OMe)pppGpC, m⁷G_(3′OMe)pppGpG,m⁷G_(3′OMe)pppGpU, m⁷G_(3′OMe)pppUpA, m⁷G_(3′OMe)pppUpC,m⁷G_(3′OMe)pppUpG, and m⁷G_(3′OMe)pppUpU.

In some embodiments, a trinucleotide cap comprises m⁷G_(3′OMe)pppApA. Insome embodiments, a trinucleotide cap comprises m⁷G_(3′OMe)pppApC. Insome embodiments, a trinucleotide cap comprises m⁷G_(3′OMe)pppApG. Insome embodiments, a trinucleotide cap comprises m⁷G_(3′OMe)pppApU. Insome embodiments, a trinucleotide cap comprises m⁷G_(3′OMe)pppCpA. Insome embodiments, a trinucleotide cap comprises m⁷G_(3′OMe)pppCpC. Insome embodiments, a trinucleotide cap comprises m⁷G_(3′OMe)pppCpG. Insome embodiments, a trinucleotide cap comprises m⁷G_(3′OMe)pppCpU. Insome embodiments, a trinucleotide cap comprises m⁷G_(3′OMe)pppGpA. Insome embodiments, a trinucleotide cap comprises m⁷G_(3′OMe)pppGpC. Insome embodiments, a trinucleotide cap comprises m⁷G_(3′OMe)pppGpG. Insome embodiments, a trinucleotide cap comprises m⁷G_(3′OMe)pppGpU. Insome embodiments, a trinucleotide cap comprises m⁷G_(3′OMe)pppUpA. Insome embodiments, a trinucleotide cap comprises m⁷G_(3′OMe)pppUpC. Insome embodiments, a trinucleotide cap comprises m⁷G_(3′OMe)pppUpG. Insome embodiments, a trinucleotide cap comprises m⁷G_(3′OMe)pppUpU.

A trinucleotide cap, in other embodiments, comprises a sequence selectedfrom the following sequences: m⁷G_(3′OMe)pppA_(2′OMe)pA,m⁷G_(3′OMe)pppA_(2′OMe)pC, m⁷G_(3′OMe)pppA_(2′OMe)pG,m⁷G_(3′OMe)pppA_(2′OMe)pU, m⁷G_(3′OMe)pppC_(2′OMe)pA,m⁷G_(3′OMe)pppC_(2′OMe)pC, m⁷G_(3′OMe)pppC_(2′OMe)pG,m⁷G_(3′OMe)pppC_(2′OMe)pU, m⁷G_(3′OMe)pppG_(2′OMe)pA,M7G_(3′OMe)pppG_(2′OMe)pC, m⁷G_(3′OMe)pppG_(2′OMe)pG,m⁷G_(3′OMe)pppG_(2′OMe)pU, m⁷G_(3′OMe)pppU_(2′OMe)pA,m⁷G_(3′OMe)pppU_(2′OMe)pC, m⁷G_(3′OMe)pppU_(2′OMe)pG, andm⁷G_(3′OMe)pppU_(2′OMe)pU.

In some embodiments, a trinucleotide cap comprisesm⁷G_(3′OMe)pppA_(2′OMe)pA. In some embodiments, a trinucleotide capcomprises m⁷G_(3′OMe)pppA_(2′OMe)pC. In some embodiments, atrinucleotide cap comprises m⁷G_(3′OMe)pppA_(2′OMe)pG. In someembodiments, a trinucleotide cap comprises m⁷G_(3′OMe)pppA_(2′OMe)pU. Insome embodiments, a trinucleotide cap comprisesm⁷G_(3′OMe)pppC_(2′OMe)pA. In some embodiments, a trinucleotide capcomprises m⁷G_(3′OMe)pppC_(2′OMe)pC. In some embodiments, atrinucleotide cap comprises m⁷G_(3′OMe)pppC_(2′OMe)pG. In someembodiments, a trinucleotide cap comprises m⁷G_(3′OMe)pppC_(2′OMe)pU. Insome embodiments, a trinucleotide cap comprisesm⁷G_(3′OMe)pppG_(2′OMe)pA. In some embodiments, a trinucleotide capcomprises m⁷G_(3′OMe)pppG_(2′OMe)pC. In some embodiments, atrinucleotide cap comprises m⁷G_(3′OMe)pppG_(2′OMe)pG. In someembodiments, a trinucleotide cap comprises m⁷G_(3′OMe)pppG_(2′OMe)pU. Insome embodiments, a trinucleotide cap comprisesm⁷G_(3′OMe)pppU_(2′OMe)pA. In some embodiments, a trinucleotide capcomprises m⁷G_(3′OMe)pppU_(2′OMe)pC. In some embodiments, atrinucleotide cap comprises m⁷G_(3′OMe)pppU_(2′OMe)pG. In someembodiments, a trinucleotide cap comprises m⁷G_(3′OMe)pppU_(2′OMe)pU.

A trinucleotide cap, in still other embodiments, comprises a sequenceselected from the following sequences: m⁷GpppA_(2′OMe)pA,m⁷GpppA_(2′OMe)pC, m⁷GpppA_(2′OMe)pG, m⁷GpppA_(2′OMe)pU,m⁷GpppC_(2′OMe)pA, m⁷GpppC_(2′OMe)pC, m⁷GpppC_(2′OMe)pG,m⁷GpppC_(2′OMe)pU, m⁷GpppG_(2′OMe)pA, m⁷GpppG_(2′OMe)pC,m⁷GpppG_(2′OMe)pG, m⁷GpppG_(2′OMe)pU, m⁷GpppU_(2′OMe)pA,m⁷GpppU_(2′OMe)pC, m⁷GpppU_(2′OMe)pG, and m⁷GpppU_(2′OMe)pU.

In some embodiments, a trinucleotide cap comprises m⁷GpppA_(2′OMe)pA. Insome embodiments, a trinucleotide cap comprises m⁷GpppA_(2′OMe)pC. Insome embodiments, a trinucleotide cap comprises m⁷GpppA_(2′OMe)pG. Insome embodiments, a trinucleotide cap comprises m⁷GpppA_(2′OMe)pU. Insome embodiments, a trinucleotide cap comprises m⁷GpppC_(2′OMe)pA. Insome embodiments, a trinucleotide cap comprises m⁷GpppC_(2′OMe)pC. Insome embodiments, a trinucleotide cap comprises m⁷GpppC_(2′OMe)pG. Insome embodiments, a trinucleotide cap comprises m⁷GpppC_(2′OMe)pU. Insome embodiments, a trinucleotide cap comprises m⁷GpppG_(2′OMe)pA. Insome embodiments, a trinucleotide cap comprises m⁷GpppG_(2′OMe)pC. Insome embodiments, a trinucleotide cap comprises m⁷GpppG_(2′OMe)pG. Insome embodiments, a trinucleotide cap comprises m⁷GpppG_(2′OMe)pU. Insome embodiments, a trinucleotide cap comprises m⁷GpppU_(2′OMe)pA. Insome embodiments, a trinucleotide cap comprises m⁷GpppU_(2′OMe)pC. Insome embodiments, a trinucleotide cap comprises m⁷GpppU_(2′OMe)pG. Insome embodiments, a trinucleotide cap comprises m⁷GpppU_(2′OMe)pU.

In some embodiments, a trinucleotide cap comprises GAG. In someembodiments, a trinucleotide cap comprises GCG. In some embodiments, atrinucleotide cap comprises GUG. In some embodiments, a trinucleotidecap comprises GGG.

In Vitro Transcription Methods

Some aspects of the present disclosure provide methods of producing(synthesizing) a RNA transcript (e.g., mRNA transcript) comprisingcontacting a DNA template with a RNA polymerase (e.g., a T7 RNApolymerase such as a T7 RNA polymerase variant) under conditions thatresult in the production of RNA transcript.

In some embodiments, the methods comprise contacting a DNA template witha T7 RNA polymerase (e.g., SEQ ID NO:1, 99, or 100) variant having aS43A substitution (e.g., a S43A T7 RNAP variant or a S43A* T7 RNAPvariant) under conditions that result in the production of RNAtranscript. In other embodiments, the methods comprise contacting a DNAtemplate with a T7 RNA polymerase (e.g., SEQ ID NO:1, 99, or 100)variant having a G47A substitution (e.g., a G47A T7 RNAP variant or aG47A* T7 RNAP variant)under conditions that result in the production ofRNA transcript. In yet other embodiments, the methods comprisecontacting a DNA template with a T7 RNA polymerase (e.g., SEQ ID NO:1,99, or 100) variant having a R257A substitution (e.g., a R257A T7 RNAPvariant or a R257A* T7 RNAP variant) under conditions that result in theproduction of RNA transcript. In still other embodiments, the methodscomprise contacting a DNA template with a T7 RNA polymerase (e.g., SEQID NO:1, 99, or 100) variant having a G259A substitution (e.g., a G259AT7 RNAP variant or a G259A* T7 RNAP variant) under conditions thatresult in the production of RNA transcript.

In some embodiments, the methods comprise contacting a DNA template witha T7 RNA polymerase variant that comprises an (at least one) additionalC terminal amino acid (e.g., Gly, Ala, GlyGly, AlaAla, GlyAla, orAlaGly).

In some embodiments, the methods comprise contacting a DNA template witha T7 RNA polymerase variant that comprises a E42R, S43A, S43E, S43L,S43R, E45R, E45L, M46A, G47A, G47E, G47L, G47R, N165W, E167M, E167N,E168I, E168T, E168V, A181F, A181W, G184M, E187F, A255Q, A255K, A255I,A255Y, R257A, R257E, R257L, R257W, G259A, G259E, G259L, G259R, A260W, orA260R substitution, or any combination of two or more of the foregoingsubstitutions and optionally an additional C terminal amino acid (e.g.,Gly, Ala, GlyGly, AlaAla, GlyAla, or AlaGly).

In some aspects, the present disclosure provides methods of performingan IVT reaction, comprising contacting a DNA template with the RNApolymerase (e.g., a T7 RNA polymerase, such as a T7 RNA polymerasevariant) in the presence of nucleoside triphosphates and buffer underconditions that result in the production of RNA transcripts.

Other aspects of the present disclosure provide co-transcriptionalcapping methods that comprise reacting a polynucleotide template with aT7 RNA polymerase variant, nucleoside triphosphates, and a cap analogunder in vitro transcription reaction conditions to produce RNAtranscript.

In some embodiments, a co-transcriptional capping method for RNAsynthesis comprises reacting a polynucleotide template with (a) a T7 RNApolymerase variant comprising at least one amino acid substitution,relative to wild-type RNA polymerase, that causes at least one loopstructure of the RNA polymerase variant to undergo a conformationalchange to a helix structure as the RNA polymerase variant transitionsfrom an initiation complex to an elongation complex, (b) nucleosidetriphosphates, and (c) a trinucleotide cap comprising sequenceGpppA_(2′Ome)pG, under in vitro transcription reaction conditions toproduce RNA transcript, wherein the polynucleotide template includes a2′-deoxythymidine residue at template position +1.

IVT conditions typically require a purified linear DNA templatecontaining a promoter, nucleoside triphosphates, a buffer system thatincludes dithiothreitol (DTT) and magnesium ions, and a RNA polymerase.The exact conditions used in the transcription reaction depend on theamount of RNA needed for a specific application. Typical IVT reactionsare performed by incubating a DNA template with a RNA polymerase andnucleoside triphosphates, including GTP, ATP, CTP, and UTP (ornucleotide analogs) in a transcription buffer. A RNA transcript having a5′ terminal guanosine triphosphate is produced from this reaction.

A deoxyribonucleic acid (DNA) is simply a nucleic acid template for RNApolymerase. A DNA template may include a polynucleotide encoding apolypeptide of interest (e.g., an antigenic polypeptide). A DNAtemplate, in some embodiments, includes a RNA polymerase promoter (e.g.,a T7 RNA polymerase promoter) located 5′ from and operably linked topolynucleotide encoding a polypeptide of interest. A DNA template mayalso include a nucleotide sequence encoding a polyadenylation (polyA)tail located at the 3′ end of the gene of interest.

Polypeptides of interest include, but are not limited to, biologics,antibodies, antigens (vaccines), and therapeutic proteins. The term“protein” encompasses peptides.

A RNA transcript, in some embodiments, is the product of an IVTreaction. A RNA transcript, in some embodiments, is a messenger RNA(mRNA) that includes a nucleotide sequence encoding a polypeptide ofinterest linked to a polyA tail. In some embodiments, the mRNA ismodified mRNA (mmRNA), which includes at least one modified nucleotide.

A nucleotide includes a nitrogenous base, a five-carbon sugar (ribose ordeoxyribose), and at least one phosphate group. Nucleotides includenucleoside monophosphates, nucleoside diphosphates, and nucleosidetriphosphates. A nucleoside monophosphate (NMP) includes a nucleobaselinked to a ribose and a single phosphate; a nucleoside diphosphate(NDP) includes a nucleobase linked to a ribose and two phosphates; and anucleoside triphosphate (NTP) includes a nucleobase linked to a riboseand three phosphates. Nucleotide analogs are compounds that have thegeneral structure of a nucleotide or are structurally similar to anucleotide. Nucleotide analogs, for example, include an analog of thenucleobase, an analog of the sugar and/or an analog of the phosphategroup(s) of a nucleotide.

A nucleoside includes a nitrogenous base and a 5-carbon sugar. Thus, anucleoside plus a phosphate group yields a nucleotide. Nucleosideanalogs are compounds that have the general structure of a nucleoside orare structurally similar to a nucleoside. Nucleoside analogs, forexample, include an analog of the nucleobase and/or an analog of thesugar of a nucleoside.

It should be understood that the term “nucleotide” includesnaturally-occurring nucleotides, synthetic nucleotides and modifiednucleotides, unless indicated otherwise. Examples of naturally-occurringnucleotides used for the production of RNA, e.g., in an IVT reaction, asprovided herein include adenosine triphosphate (ATP), guanosinetriphosphate (GTP), cytidine triphosphate (CTP), uridine triphosphate(UTP), and 5-methyluridine triphosphate (m⁵UTP). In some embodiments,adenosine diphosphate (ADP), guanosine diphosphate (GDP), cytidinediphosphate (CDP), and/or uridine diphosphate (UDP) are used.

Examples of nucleotide analogs include, but are not limited to,antiviral nucleotide analogs, phosphate analogs (soluble or immobilized,hydrolyzable or non-hydrolyzable), dinucleotide, trinucleotide,tetranucleotide, e.g., a cap analog, or a precursor/substrate forenzymatic capping (vaccinia or ligase), a nucleotide labeled with afunctional group to facilitate ligation/conjugation of cap or 5′ moiety(IRES), a nucleotide labeled with a 5′ PO4 to facilitate ligation of capor 5′ moiety, or a nucleotide labeled with a functional group/protectinggroup that can be chemically or enzymatically cleaved. Examples ofantiviral nucleotide/nucleoside analogs include, but are not limited, toGanciclovir, Entecavir, Telbivudine, Vidarabine and Cidofovir.

Modified nucleotides may include modified nucleobases. For example, aRNA transcript (e.g., mRNA transcript) of the present disclosure mayinclude a modified nucleobase selected from pseudouridine (ψ),1-methylpseudouridine (m1ψ), 1-ethylpseudouridine, 2-thiouridine,4′-thiouridine, 2-thio-1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine,2-thio-dihydropseudouridine, 2-thio-dihydrouridine,2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine,4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine,4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine,5-methyluridine, 5-methoxyuridine (mo5U) and 2′-O-methyl uridine. Insome embodiments, a RNA transcript (e.g., mRNA transcript) includes acombination of at least two (e.g., 2, 3, 4 or more) of the foregoingmodified nucleobases.

The nucleoside triphosphates (NTPs) as provided herein may compriseunmodified or modified ATP, modified or unmodified UTP, modified orunmodified GTP, and/or modified or unmodified CTP. In some embodiments,NTPs of an IVT reaction comprise unmodified ATP. In some embodiments,NTPs of an IVT reaction comprise modified ATP. In some embodiments, NTPsof an IVT reaction comprise unmodified UTP. In some embodiments, NTPs ofan IVT reaction comprise modified UTP. In some embodiments, NTPs of anIVT reaction comprise unmodified GTP. In some embodiments, NTPs of anIVT reaction comprise modified GTP. In some embodiments, NTPs of an IVTreaction comprise unmodified CTP. In some embodiments, NTPs of an IVTreaction comprise modified CTP.

The concentration of nucleoside triphosphates and cap analog present inan IVT reaction may vary. In some embodiments, NTPs and cap analog arepresent in the reaction at equimolar concentrations. In someembodiments, the molar ratio of cap analog (e.g., trinucleotide cap) tonucleoside triphosphates in the reaction is greater than 1:1. Forexample, the molar ratio of cap analog to nucleoside triphosphates inthe reaction may be 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1,20:1, 25:1, 50:1, or 100:1. In some embodiments, the molar ratio of capanalog (e.g., trinucleotide cap) to nucleoside triphosphates in thereaction is less than 1:1. For example, the molar ratio of cap analog(e.g., trinucleotide cap) to nucleoside triphosphates in the reactionmay be 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25,1:50, or 1:100.

The composition of NTPs in an IVT reaction may also vary. For example,ATP may be used in excess of GTP, CTP and UTP. As a non-limitingexample, an IVT reaction may include 7.5 millimolar GTP, 7.5 millimolarCTP, 7.5 millimolar UTP, and 3.75 millimolar ATP. The same IVT reactionmay include 3.75 millimolar cap analog (e.g., trinucleotide cap). Insome embodiments, the molar ratio of G:C:U:A:cap is 1:1:1:0.5:0.5. Insome embodiments, the molar ratio of G:C:U:A:cap is 1:1:0.5:1:0.5. Insome embodiments, the molar ratio of G:C:U:A:cap is 1:0.5:1:1:0.5. Insome embodiments, the molar ratio of G:C:U:A:cap is 0.5:1:1:1:0.5.

In some embodiments, a RNA transcript (e.g., mRNA transcript) includes amodified nucleobase selected from pseudouridine (ψ),1-methylpseudouridine (m¹ψ), 5-methoxyuridine (mo⁵U), 5-methylcytidine(m⁵C), α-thio-guanosine and α-thio-adenosine. In some embodiments, a RNAtranscript (e.g., mRNA transcript) includes a combination of at leasttwo (e.g., 2, 3, 4 or more) of the foregoing modified nucleobases.

In some embodiments, a RNA transcript (e.g., mRNA transcript) includespseudouridine (ψ). In some embodiments, a RNA transcript (e.g., mRNAtranscript) includes 1-methylpseudouridine (m¹ψ). In some embodiments, aRNA transcript (e.g., mRNA transcript) includes 5-methoxyuridine (mo⁵U).In some embodiments, a RNA transcript (e.g., mRNA transcript) includes5-methylcytidine (m⁵C). In some embodiments, a RNA transcript (e.g.,mRNA transcript) includes α-thio-guanosine. In some embodiments, a RNAtranscript (e.g., mRNA transcript) includes α-thio-adenosine.

In some embodiments, the polynucleotide (e.g., RNA polynucleotide, suchas mRNA polynucleotide) is uniformly modified (e.g., fully modified,modified throughout the entire sequence) for a particular modification.For example, a polynucleotide can be uniformly modified with1-methylpseudouridine (m¹ψ), meaning that all uridine residues in themRNA sequence are replaced with 1-methylpseudouridine (m¹ψ). Similarly,a polynucleotide can be uniformly modified for any type of nucleosideresidue present in the sequence by replacement with a modified residuesuch as any of those set forth above. Alternatively, the polynucleotide(e.g., RNA polynucleotide, such as mRNA polynucleotide) may not beuniformly modified (e.g., partially modified, part of the sequence ismodified). Each possibility represents a separate embodiment of thepresent invention.

In some embodiments, the buffer system contains tris. The concentrationof tris used in an IVT reaction, for example, may be at least 10 mM, atleast 20 mM, at least 30 mM, at least 40 mM, at least 50 mM, at least 60mM, at least 70 mM, at least 80 mM, at least 90 mM, at least 100 mM orat least 110 mM phosphate. In some embodiments, the concentration ofphosphate is 20-60 mM or 10-100 mM.

In some embodiments, the buffer system contains dithiothreitol (DTT).The concentration of DTT used in an IVT reaction, for example, may be atleast 1 mM, at least 5 mM, or at least 50 mM. In some embodiments, theconcentration of DTT used in an IVT reaction is 1-50 mM or 5-50 mM. Insome embodiments, the concentration of DTT used in an IVT reaction is 5mM.

In some embodiments, the buffer system contains magnesium. In someembodiments, the molar ratio of NTP to magnesium ions (Mg²⁺; e.g.,MgCl₂) present in an IVT reaction is 1:1 to 1:5. For example, the molarratio of NTP to magnesium ions may be 1:1, 1:2, 1:3, 1:4 or 1:5.

In some embodiments, the molar ratio of NTP plus cap analog (e.g.,tricnucleotide cap, such as GAG) to magnesium ions (Mg²⁺; e.g., MgCl₂)present in an IVT reaction is 1:1 to 1:5. For example, the molar ratioof NTP+trinucleotide cap (e.g., GAG) to magnesium ions may be 1:1, 1:2,1:3, 1:4 or 1:5.

In some embodiments, the buffer system contains Tris-HCl, spermidine(e.g., at a concentration of 1-30 mM), TRITON® X-100 (polyethyleneglycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether) and/or polyethyleneglycol (PEG).

The addition of nucleoside triphosphates (NTPs) to the 3′ end of agrowing RNA strand is catalyzed by a polymerase, such as T7 RNApolymerase, for example, any one or more of the T7 RNA polymerasevariants (e.g., S43A and/or G47A) of the present disclosure. In someembodiments, the RNA polymerase (e.g., T7 RNA polymerase variant) ispresent in a reaction (e.g., an IVT reaction) at a concentration of 0.01mg/ml to 1 mg/ml. For example, the RNA polymerase may be present in areaction at a concentration of 0.01 mg/mL, 0.05 mg/ml, 0.1 mg/ml, 0.5mg/ml or 1.0 mg/ml.

Surprisingly, use of the combination of a T7 RNAP variant (e.g., E42,S43, Y44, E45, M46, G47, A255, R257, or G259, e.g., S43A or G47A) asprovided herein with a cap analog (e.g., GpppA_(2′Ome)pG), in an invitro transcription reaction, for example, results in the production ofRNA transcript, wherein greater than 80% of the RNA transcript producedincludes a functional cap. In some embodiments, greater than 85% of theRNA transcript produced includes a functional cap. In some embodiments,greater than 90% of the RNA transcript produced includes a functionalcap. In some embodiments, greater than 95% of the RNA transcriptproduced includes a functional cap. In some embodiments, greater than96% of the RNA transcript produced includes a functional cap. In someembodiments, greater than 97% of the RNA transcript produced includes afunctional cap. In some embodiments, greater than 98% of the RNAtranscript produced includes a functional cap. In some embodiments,greater than 99% of the RNA transcript produced includes a functionalcap.

Also surprising was the finding that use of a polynucleotide templatethat includes a 2′-deoxythymidine residue or 2′-deoxycytidine residue attemplate position +1 results in the production of RNA transcript,wherein greater than 80% (e.g., greater than 85%, greater than 90%, orgreater than 95%) of the RNA transcript produced includes a functionalcap. Thus, in some embodiments, a polynucleotide (e.g., DNA) templateused, for example, in an IVT reaction, includes a 2′-deoxythymidineresidue at template position +1. In other embodiments, a polynucleotide(e.g., DNA) template used, for example, in an IVT reaction, includes a2′-deoxycytidine residue at template position +1.

Applications

The RNA transcripts produced according to the present disclosure includemRNA (including modified mRNA and/or unmodified RNA), lncRNA,self-replicating RNA, circular RNA, CRISPR guide RNA, and the like. Inembodiments, the RNA is RNA (e.g., mRNA or self-replicating RNA) thatencodes a polypeptide (e.g., a therapeutic polypeptide). Thus, the RNAtranscripts produced using RNA polymerase variants of the presentdisclosure may be used in a myriad of applications.

For example, the RNA transcripts may be used to produce polypeptides ofinterest, e.g., therapeutic proteins, vaccine antigen, and the like. Insome embodiments, the RNA transcripts are therapeutic RNAs. Atherapeutic mRNA is an mRNA that encodes a therapeutic protein (the term‘protein’ encompasses peptides). Therapeutic proteins mediate a varietyof effects in a host cell or in a subject to treat a disease orameliorate the signs and symptoms of a disease. For example, atherapeutic protein can replace a protein that is deficient or abnormal,augment the function of an endogenous protein, provide a novel functionto a cell (e.g., inhibit or activate an endogenous cellular activity, oract as a delivery agent for another therapeutic compound (e.g., anantibody-drug conjugate). Therapeutic mRNA may be useful for thetreatment of the following diseases and conditions: bacterialinfections, viral infections, parasitic infections, cell proliferationdisorders, genetic disorders, and autoimmune disorders. Other diseasesand conditions are encompassed herein.

A protein of interest encoded by an mRNA as provided herein can beessentially any protein. In some embodiments, the therapeutic protein isa cytokine, a growth factor, an antibody or a fusion protein.Non-limiting examples of therapeutic proteins include blood factors(such as Factor VIII and Factor VII), complement factors, Low DensityLipoprotein Receptor (LDLR) and MUT1. Non-limiting examples of cytokinesinclude interleukins, interferons, chemokines, lymphokines and the like.Non-limiting examples of growth factors include erythropoietin, EGFs,PDGFs, FGFs, TGFs, IGFs, TNFs, CSFs, MCSFs, GMCSFs and the like.Non-limiting examples of antibodies include adalimumab, infliximab,rituximab, ipilimumab, tocilizumab, canakinumab, itolizumab,tralokinumab. Non-limiting examples of fusion proteins include, forexample, etanercept, abatacept and belatacept.

In some embodiments, the protein of interest is human erythropoietin,LDLR (for use in inhibiting cholesterol), or MUT1 (for use in thetreatment of methylmalonic acidemia (MMA)). In other embodiments, theprotein of interest encoded by the mRNA is a therapeutic antibody,including but not limited to the antibodies listed above.

A RNA transcript produced using a RNA polymerase variant as disclosedherein may encode one or more biologics. A biologic is apolypeptide-based molecule that may be used to treat, cure, mitigate,prevent, or diagnose a serious or life-threatening disease or medicalcondition. Biologics include, but are not limited to, allergenicextracts (e.g. for allergy shots and tests), blood components, genetherapy products, human tissue or cellular products used intransplantation, vaccines, monoclonal antibodies, cytokines, growthfactors, enzymes, thrombolytics, and immunomodulators, among others.

One or more biologics currently being marketed or in development may beencoded by the RNA of the present invention. While not wishing to bebound by theory, it is believed that incorporation of the encodingpolynucleotides of a known biologic into the RNA of the presentdisclosure will result in improved therapeutic efficacy due at least inpart to the specificity, purity and/or selectivity of the constructdesigns.

A RNA transcript produced using a RNA polymerase variant as disclosedherein may encode one or more antibodies. The term “antibody” includesmonoclonal antibodies (including full length antibodies which have animmunoglobulin Fc region), antibody compositions with polyepitopicspecificity, multispecific antibodies (e.g., bispecific antibodies,diabodies, and single-chain molecules), as well as antibody fragments.The term “immunoglobulin” (Ig) is used interchangeably with “antibody”herein. A monoclonal antibody is an antibody obtained from a populationof substantially homogeneous antibodies, i.e., the individual antibodiescomprising the population are identical except for possible naturallyoccurring mutations and/or post-translation modifications (e.g.,isomerizations, amidations) that may be present in minor amounts.Monoclonal antibodies are highly specific, being directed against asingle antigenic site.

Monoclonal antibodies specifically include chimeric antibodies(immunoglobulins) in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is(are) identicalwith or homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity. Chimeric antibodies include, but are notlimited to, “primatized” antibodies comprising variable domainantigen-binding sequences derived from a non-human primate (e.g., OldWorld Monkey, Ape etc.) and human constant region sequences.

Antibodies encoded in the RNA of the present disclosure may be utilizedto treat conditions or diseases in many therapeutic areas such as, butnot limited to, blood, cardiovascular, CNS, poisoning (includingantivenoms), dermatology, endocrinology, gastrointestinal, medicalimaging, musculoskeletal, oncology, immunology, respiratory, sensory andanti-infective.

A RNA transcript produced using a RNA polymerase variant as disclosedherein may encode one or more vaccine antigens. A vaccine antigen is abiological preparation that improves immunity to a particular disease orinfectious agent. One or more vaccine antigens currently being marketedor in development may be encoded by the RNA of the present disclosure.Vaccine antigens encoded in the RNA may be utilized to treat conditionsor diseases in many therapeutic areas such as, but not limited to,cancer, allergy and infectious disease. In some embodiments, a cancervaccine may be a personalized cancer vaccine in the form of a concatemeror individual RNAs encoding peptide epitopes or a combination thereof.

A RNA transcript produced using a RNA polymerase variant as disclosedherein may be designed to encode on or more antimicrobial peptides (AMP)or antiviral peptides (AVP). AMPs and AVPs have been isolated anddescribed from a wide range of animals such as, but not limited to,microorganisms, invertebrates, plants, amphibians, birds, fish, andmammals. The anti-microbial polypeptides may block cell fusion and/orviral entry by one or more enveloped viruses (e.g., HIV, HCV). Forexample, the anti-microbial polypeptide can comprise or consist of asynthetic peptide corresponding to a region, e.g., a consecutivesequence of at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or60 amino acids of the transmembrane subunit of a viral envelope protein,e.g., HIV-1 gp120 or gp41. The amino acid and nucleotide sequences ofHIV-1 gp120 or gp41 are described in, e.g., Kuiken et al., (2008). “HIVSequence Compendium,” Los Alamos National Laboratory.

In some embodiments, RNA transcripts are used as radiolabeled RNAprobes. In some embodiments, RNA transcripts are used for non-isotopicRNA labeling. In some embodiments, RNA transcripts are used as guide RNA(gRNA) for gene targeting. In some embodiments, RNA transcripts (e.g.,mRNA) are used for in vitro translation and micro injection. In someembodiments, RNA transcripts are used for RNA structure, processing andcatalysis studies. In some embodiments, RNA transcripts are used for RNAamplification. In some embodiments, RNA transcripts are used asanti-sense RNA for gene expression experiment. Other applications areencompassed by the present disclosure.

Compositions

The T7 RNAP variants of the present disclosure, in some embodiments whenused in combination with a cap analog, such as a trinucleotide cap, inan IVT reaction produces RNA that does not induce a detectable cytokineresponse, even in the absence of post-IVT purification. Thus, providedherein, in some embodiments, are composition comprising IVT RNA and apharmaceutically acceptable excipient, wherein the composition issubstantially free (e.g., does not comprise, or comprises less than 10%,less than 1%, less than 0.1%, or less than 0.01%) of cytokine-inducingRNA contaminant in the absence of post-IVT purification.

As described elsewhere herein, the T7 RNAP variants and methods of thepresent disclosure produce RNA transcript, wherein at least 80% (e.g.,80%-90%, 80-95%, 90-95%, 80-99%, 90-99%, or 90-100%) of the transcriptincludes a functional cap. Thus, also provided herein are compositioncomprising an IVT RNA and a pharmaceutically acceptable excipient,wherein the composition comprises less than 20% (e.g., 5-15%, 5-10%,1-15%, or 1-10%) uncapped RNA species. In some embodiments, thecomposition comprises less than 15% (e.g., 5-10%, 1-10%, or 1-5%)uncapped RNA species. In some embodiments, the composition comprisesless than 10% uncapped RNA species. In some embodiments, the compositioncomprises less than 5% uncapped RNA species.

In some embodiments, greater than 80% of the IVT RNA includes afunctional cap. In some embodiments, greater than 85% of the IVT RNAincludes a functional cap. In some embodiments, greater than 90% of theIVT RNA includes a functional cap. In some embodiments, greater than 95%of the IVT RNA includes a functional cap.

The IVT RNA, in some embodiments, is not chemically modified, while inother embodiments, the IVT RNA is chemically modified.

In some embodiments, unexpectedly, greater than 80% of the IVT RNAcomprises single-stranded full-length transcripts. For example, greaterthan 85% of the IVT RNA may comprise single-stranded full-lengthtranscripts. In some embodiments, greater than 90% of the IVT RNAcomprises single-stranded full-length transcripts. greater than 95% ofthe IVT RNA comprises single-stranded full-length transcripts.

In some embodiments, the RNA is produced by a process comprisingreacting a polynucleotide template with (a) a T7 RNA polymerase variantcomprising at least one amino acid substitution, relative to wild-typeRNA polymerase, that causes at least one loop structure of the RNApolymerase variant to undergo a conformational change to a helixstructure as the RNA polymerase variant transitions from an initiationcomplex to an elongation complex, (b) nucleoside triphosphates, and (c)a trinucleotide cap comprising sequence GpppA_(2′Ome)pG, under in vitrotranscription reaction conditions to produce RNA transcript, wherein thepolynucleotide template includes a 2′-deoxythymidine residue at templateposition +1.

Kits

Also provided herein are kits, such as in vitro transcription kitscomprising a RNA polymerase variant of the present disclosure. The kitsmay comprise any one or more (at least one) IVT component describedherein and any one or more (at least one) RNA polymerase variant. Forexample, a kit may include a buffer system, NTPs and a T7 RNA polymerasevariant having an amino acid sequence of SEQ ID NO:1, 99, or 100 havingat least one (or at least two, or at least three) amino acidsubstitution at position E42, S43, Y44, E45, M46, G47, A255, R257, A258,G259, A260, L261 and/or A262 and optionally at least one amino acidaddition on the C-terminal end.

Additional Embodiments

Additional embodiments of the present disclosure are encompassed by thefollowing numbered paragraphs:

1. A ribonucleic acid (RNA) polymerase variant comprising at least oneamino acid substitution, relative to wild-type RNA polymerase, thatcauses at least one loop structure of the RNA polymerase variant toundergo a conformational change to a helix structure as the RNApolymerase variant transitions from an initiation complex to anelongation complex.2. The RNA polymerase variant of paragraph 1, wherein the at least oneamino acid substitution has a higher helix propensity, relative towild-type amino acid.3. The RNA polymerase variant of paragraph 1 or 2, wherein the RNApolymerase is a T7 RNA polymerase.4. The RNA polymerase variant of any one of paragraphs 1-3, wherein theat least one loop structure is in a C-helix structure.5. The RNA polymerase variant of any one of paragraphs 1-4, wherein theat least one loop structure is in a C-linker structure.6. The RNA polymerase variant of any one of paragraphs 1-4, wherein theat least one amino acid substitution is at least one high-helixpropensity amino acid substitution.7. The RNA polymerase variant of paragraph 6, wherein the at least onehigh-helix propensity acid substitution is selected from alanine,isoleucine, leucine, methionine, lysine, glutamine, and/or glutamate.8. The RNA polymerase variant of paragraph 7, wherein the at least onehigh-helix propensity amino acid substitution is alanine.9. The RNA polymerase variant of any one of paragraphs 4-8, wherein theT7 RNA polymerase comprising an amino acid sequence identified by SEQ IDNO:1, SEQ ID NO:99, or SEQ ID NO:100 modified to include at least oneamino acid substitution of a high-helix propensity amino acid at aposition selected from E42, S43, Y44, E45, M46, G47, R257, and G259.10. The RNA polymerase variant of paragraph 9, wherein that at least oneamino acid substitution comprises S43A.11. The RNA polymerase variant of paragraph 9, wherein that at least oneamino acid substitution comprises G47A.12. The RNA polymerase variant of any one of paragraphs 5-8, wherein theT7 RNA polymerase comprising an amino acid sequence identified by SEQ IDNO:1, SEQ ID NO:99, or SEQ ID NO:100 modified to include at least oneamino acid substitution of a high-helix propensity amino acid at aposition selected from R257, A258, G259, A260, L261 and A262.13. The RNA polymerase variant of paragraph 12, wherein the at least oneamino acid substitution comprises R257A.14. The RNA polymerase variant of paragraph 12, wherein the at least oneamino acid substitution comprises G259A.15. A T7 ribonucleic acid (RNA) polymerase comprising an amino acidsequence identified by SEQ ID NO:1, SEQ ID NO:99, or SEQ ID NO:100modified to include an amino acid substitution of a high-helixpropensity amino acid at position G47, S43, R257, or G259.16. The T7 RNA polymerase of paragraph 15, wherein the high-helixpropensity amino acid is selected from alanine, isoleucine, leucine,methionine, lysine, glutamine, and/or glutamate.17. The T7 RNA polymerase of paragraph 16, wherein the high-helixpropensity amino acid is alanine.18. A T7 RNA polymerase comprising an amino acid sequence identified bySEQ ID NO:2, SEQ ID NO:107, or SEQ ID NO:108.19. A T7 RNA polymerase comprising an amino acid sequence identified bySEQ ID NO:3, SEQ ID NO:109, or SEQ ID NO:110.20. A T7 RNA polymerase comprising an amino acid sequence identified bySEQ ID NO:4, SEQ ID NO:111, or SEQ ID NO:112.21. A T7 RNA polymerase comprising an amino acid sequence identified bySEQ ID NO:5, SEQ ID NO:113, or SEQ ID NO:114.22. A method of producing a ribonucleic acid (RNA) comprising contactinga DNA template with the RNA polymerase of any one of paragraphs 1-21under conditions that result in the production of RNA transcript.23. A method of performing an in vitro transcription (IVT) reaction,comprising contacting a DNA template with the RNA polymerase of any oneof paragraphs 1-21 in the presence of nucleoside triphosphates andbuffer under conditions that result in the production of RNAtranscripts.24. The method of paragraph 23, wherein the RNA transcript produced,when delivered to cells, optionally in unpurified form, stimulatescytokine response that is at least 50% lower relative to RNA producedusing wild-type RNA polymerase.25. The method of any one of paragraphs 23-24, wherein the concentrationof double-stranded RNA transcript produced by IVT is at least 50% lowerrelative to dsRNA transcript produced using wild-type polymerase.26. The method of any one of paragraphs 23-25, wherein less than 20% ofthe RNA transcripts produced exhibit 3′ heterogeneity.27. The method of any one of paragraphs 23-26, wherein less than 50% ofthe RNA transcript produced is truncated RNA transcript.28. The method of any one of paragraphs 23-28, wherein less than 50% ofthe RNA transcript produced is run-on RNA transcript.29. The method of any one of paragraphs 23-28, wherein the amount offull-length RNA transcript produced is at least 15 times greater thanthe amount of the DNA template30. The method of any one of paragraphs 23-29, wherein the ratio oftruncated RNA transcript:full-length RNA transcript produced is lessthan 1:1.31. The method of any one of paragraphs 23-30, wherein the RNAtranscript produced has less than 1 mutation per 100 nucleotidesrelative to the DNA template.32. A nucleic acid encoding the RNA polymerase of any one of paragraphs1-21.33. A vector comprising the nucleic acid of paragraph 33.34. A host cell comprising the nucleic acid of paragraph 33 or thevector of paragraph 34.35. A kit comprising the RNA polymerase of any one of paragraphs 1-21.36. A composition comprising the RNA polymerase of any one of paragraphs1-21.37. A ribonucleic acid (RNA) produced by the method of any one ofparagraphs 22-31.38. The RNA of paragraph 37 formulated in a lipid nanoparticle.39. The RNA of paragraph 38, wherein the lipid nanoparticle comprises amolar ratio of 20-60% ionizable amino lipid, 5-25% non-cationic lipid,25-55% sterol, and 0.5-15% PEG-modified lipid.40. A co-transcriptional capping method for ribonucleic acid (RNA)synthesis, the method comprising reacting a polynucleotide template witha T7 RNA polymerase variant, nucleoside triphosphates, and a cap analogunder in vitro transcription reaction conditions to produce RNAtranscript.41. The method of paragraph 40, wherein greater than 80%, greater than85%, or greater than 90% of the RNA transcript produced includes afunctional cap.42. The method of paragraph 41, wherein greater than 95% of the RNAtranscript produced includes a functional cap.43. The method of any one of paragraphs 40-42, wherein the nucleosidetriphosphates comprise unmodified or modified ATP, modified orunmodified UTP, modified or unmodified GTP, and/or modified orunmodified CTP.44. The method of any one of paragraphs 40-43, wherein the T7 RNApolymerase variant is a T7 polymerase variant of any one of paragraph1-21.45. The method of paragraph 44, wherein the T7 polymerase variantcomprises an amino acid sequence identified by SEQ ID NO:1, SEQ IDNO:99, or SEQ ID NO:100 modified to include at least one amino acidsubstitution of a high-propensity amino acid at a position selected fromE42, S43, Y44, E45, M46, G47, R257, and G259.46. The method of any one of paragraphs 40-45, the T7 polymerase variantcomprises an amino acid sequence identified by SEQ ID NO:1, SEQ IDNO:99, or SEQ ID NO:100 modified to include amino acid substitution ofG47A.47. The method of any one of paragraphs 40-45, the T7 polymerase variantcomprises an amino acid sequence identified by SEQ ID NO:1, SEQ IDNO:99, or SEQ ID NO:100 modified to include amino acid substitution ofS43A.48. The method of any one of paragraphs 40-47, wherein the nucleosidetriphosphates and cap analog are present in the reaction at equimolarconcentrations.49. The method of any one of paragraphs 40-47, wherein a molar ratio ofcap analog to nucleoside triphosphates in the reaction is greater than1:1.50. The method of any one of paragraphs 40-47, wherein a molar ratio ofcap analog to nucleoside triphosphates in the reaction is less than 1:1.51. The method of any one of paragraphs 40-50, wherein the cap analog isa dinucleotide cap, a trinucleotide cap, or a tetranucleotide cap.52. The method of any one of paragraphs 40-50, wherein the cap analog isa trinucleotide cap.53. The method of paragraph 51, wherein the trinucleotide cap comprisesa sequence selected from the following sequences: GAA, GAC, GAG, GAU,GCA, GCC, GCG, GCU, GGA, GGC, GGG, GGU, GUA, GUC, GUG, and GUU.54. The method of paragraph 53, wherein the trinucleotide cap comprisesa sequence selected from the following sequences: m⁷GpppApA, m⁷GpppApC,m⁷GpppApG, m⁷GpppApU, m⁷GpppCpA, m⁷GpppCpC, m⁷GpppCpG, m⁷GpppCpU,m⁷GpppGpA, m⁷GpppGpC, m⁷GpppGpG, m⁷GpppGpU, m⁷GpppUpA, m⁷GpppUpC,m⁷GpppUpG, and m⁷GpppUpU.55. The method of paragraph 53, wherein the trinucleotide cap comprisesa sequence selected from the following sequences: m⁷G_(3′OMe)pppApA,m⁷G_(3′OMe)pppApC, m⁷G_(3′OMe)pppApG, m⁷G_(3′OMe)pppApU,m⁷G_(3′OMe)pppCpA, m⁷G_(3′OMe)pppCpC, m⁷G_(3′OMe)pppCpG,m⁷G_(3′OMe)pppCpU, m⁷G_(3′OMe)pppGpA, m⁷G_(3′OMe)pppGpC,m⁷G_(3′OMe)pppGpG, m⁷G_(3′OMe)pppGpU, m⁷G_(3′OMe)pppUpA,m⁷G_(3′OMe)pppUpC, m⁷G_(3′OMe)pppUpG, and m⁷G_(3′OMe)pppUpU.56. The method of paragraph 53, wherein the trinucleotide cap comprisesa sequence selected from the following sequences:m⁷G_(3′OMe)pppA_(2′OMe)pA, m⁷G_(3′OMe)pppA_(2′OMe)pC,m⁷G_(3′OMe)pppA_(2′OMe)pG, m⁷G_(3′OMe)pppA_(2′OMe)pU,m⁷G_(3′OMe)pppC_(2′OMe)pA, m⁷G_(3′OMe)pppC_(2′OMe)pC,m⁷G_(3′OMe)pppC_(2′OMe)pG, m⁷G_(3′OMe)pppC_(2′OMe)pU,m⁷G_(3′OMe)pppG_(2′OMe)pA, m⁷G_(3′OMe)pppG_(2′OMe)pC,m⁷G_(3′OMe)pppG_(2′OMe)pG, m⁷G_(3′OMe)pppG_(2′OMe)pU,m⁷G_(3′OMe)pppU_(2′OMe)pA, m⁷G_(3′OMe)pppU_(2′OMe)pC,m⁷G_(3′OMe)pppU_(2′OMe)pG, and m⁷G_(3′OMe)pppU_(2′OMe)pU.57. The method of paragraph 53, wherein the trinucleotide cap comprisesa sequence selected from the following sequences: m⁷GpppA_(2′OMe)pA,m⁷GpppA_(2′OMe)pC, m⁷GpppA_(2′OMe)pG, m⁷GpppA_(2′OMe)pU,m⁷GpppC_(2′OMe)pA, m⁷GpppC_(2′OMe)pC, m⁷GpppC_(2′OMe)pG,m⁷GpppC_(2′OMe)pU, m⁷GpppG_(2′OMe)pA, m⁷GpppG_(2′OMe)pC,m⁷GpppG_(2′OMe)pG, m⁷GpppG_(2′OMe)pU, m⁷GpppU_(2′OMe)pA,m⁷GpppU_(2′OMe)pC, m⁷GpppU_(2′OMe)pG, and m⁷GpppU_(2′OMe)pU.58. The method of any one of paragraphs 53-57, wherein the trinucleotidecap comprises a sequence selected from the following sequences: GAG,GCG, GUG, and GGG.59. The method of paragraph 58, wherein the trinucleotide cap comprisessequence GAG.60. The method of paragraph 59, wherein the trinucleotide cap comprisesGpppA_(2′OMe)pG.61. The method of any one of paragraphs 40-60, wherein thepolynucleotide template includes a 2′-deoxythymidine residue at templateposition +1.62. The method of any one of paragraphs 40-60, wherein thepolynucleotide template includes a 2′-deoxycytidine residue at templateposition +1.63. A co-transcriptional capping method for RNA synthesis, the methodcomprising reacting a polynucleotide template with (a) a T7 RNApolymerase variant comprising at least one amino acid substitution,relative to wild-type RNA polymerase, that causes at least one loopstructure of the RNA polymerase variant to undergo a conformationalchange to a helix structure as the RNA polymerase variant transitionsfrom an initiation complex to an elongation complex, (b) nucleosidetriphosphates, and (c) a trinucleotide cap comprising sequenceGpppA_(2′OMe)pG, under in vitro transcription reaction conditions toproduce RNA transcript, wherein the polynucleotide template includes a2′-deoxythymidine residue at template position +1.64. The method of any one of paragraph 40-63, wherein the RNA transcriptproduced, when delivered to cells, optionally in unpurified form, doesnot stimulate a detectable cytokine response.65. A composition comprising an in vitro-transcribed (IVT) RNA and apharmaceutically acceptable excipient, wherein the composition issubstantially free of cytokine-inducing RNA contaminant in the absenceof post-IVT purification.66. A composition comprising an in vitro-transcribed (IVT) RNA and apharmaceutically acceptable excipient, wherein the composition has lessthan 5% uncapped RNA species.67. The composition of paragraph 65 or 66, wherein greater than 80%,85%, or 90% of the IVT RNA include a functional cap.68. The composition of paragraph 67, wherein greater than 95% of the IVTRNA include a functional cap.69. The composition of any one of paragraphs 65-68, wherein the IVT RNAis not chemically modified.70. The composition of any one of paragraphs 65-68, wherein the IVT RNAis chemically modified.71. The composition of any one of paragraphs 65-70, wherein greater than95% of the IVT RNA comprises single-stranded full-length transcripts.72. The composition of any one of paragraphs 65-71, wherein the RNA isproduced by a process comprising:

reacting a polynucleotide template with (a) a T7 RNA polymerase variantcomprising at least one amino acid substitution, relative to wild-typeRNA polymerase, that causes at least one loop structure of the RNApolymerase variant to undergo a conformational change to a helixstructure as the RNA polymerase variant transitions from an initiationcomplex to an elongation complex, (b) nucleoside triphosphates, and (c)a trinucleotide cap comprising sequence GpppA_(2′Ome)pG, under in vitrotranscription reaction conditions to produce RNA transcript, wherein thepolynucleotide template includes a 2′-deoxythymidine residue at templateposition +1.

73. A T7 RNA polymerase comprising at least one additional C-terminalamino acid relative to wild-type T7 RNA polymerase.74. The T7 RNA polymerase of paragraph 73 comprising at least twoadditional C-terminal amino acids.75. The T7 RNA polymerase of paragraph 74, wherein the at least twoadditional C-terminal amino acids comprise the same type of amino acidor at least two different types of amino acids.76. The T7 RNA polymerase of any one of paragraphs 73-75 comprising 1 to10 additional C-terminal amino acids.77. The T7 RNA polymerase of paragraph 76 comprising 1 to 5 additionalC-terminal amino acids.78. The T7 RNA polymerase of any one of paragraphs 73-77, wherein the T7RNA polymerase comprises a C terminus that comprises a FAFAX_(n) (SEQ IDNO; 171) motif, wherein X is any amino acid and n is any integer greaterthan zero.79. The T7 RNA polymerase of paragraph 78, wherein X is G or A, andoptionally wherein X_(n) is GG or AA.80. The T7 RNA polymerase of paragraph 78 or 79, wherein n is 1, 2, 3,4, or 5.81. A T7 RNA polymerase comprising a C terminus that comprises a FAFAG(SEQ ID NO: 329) motif.82. A T7 RNA polymerase comprising a C terminus that comprises aXAFAX_(n) motif, a FXFAX_(n) motif, FAXAX_(n) motif, or a FAFXX_(n)motif, wherein each X is any amino acid and n is any integer greaterthan zero.83. The T7 RNA polymerase of any one of paragraphs 73-82, wherein the T7RNA polymerase comprises at least one substitution at a positioncorresponding to position S43, G47, R257, or G259 of a wild-type T7 RNApolymerase, and optionally at least one additional amino acidsubstitution.84. The T7 RNA polymerase of paragraph 83, wherein the wild-type T7 RNApolymerase comprises an amino acid sequence identified by SEQ ID NO:1.85. A T7 RNA polymerase comprising an amino acid sequence of SEQ IDNO:99 modified to include at least one substitution selected from G47,S43, R257, and G259, and optionally at least one additional amino acidsubstitution.86. A T7 RNA polymerase comprising an amino acid sequence of any one ofSEQ ID NOs:100, 294, 296, or 296 modified to include at least onesubstitution selected from G47, S43, R257, and G259, and optionally atleast one additional amino acid substitution.87. The T7 RNA polymerase of any one of paragraphs 83-86, comprising atleast one substitution selected from S43A, G47A, R257A, and G259A.88. A T7 RNA polymerase comprising an amino acid sequence of any one ofSEQ ID NOs: 294-313, wherein x is any amino acid and n is any integer,e.g., between 1 and 5 (e.g., 1, 2, 3, 4, or 5), and optionally whereinthe T7 RNA polymerase further comprises at least one additional aminoacid substitution.89. A method of performing an in vitro transcription (IVT) reaction,comprising contacting a DNA template with the RNA polymerase of any oneof paragraphs 73-88 in the presence of nucleoside triphosphates andbuffer under conditions that result in the production of RNAtranscripts.90. The method of paragraph 89, wherein the RNA produced, when deliveredto cells stimulates a cytokine response that is at least 50% lowerrelative to the dsRNA transcript produced using a WT T7 RNAP.91. The method of paragraph 90, wherein the RNA is delivered to thecells in unpurified form.92. The method of any one of paragraphs 89-92, wherein less than 30% ofthe RNA transcripts produced exhibit 3′ heterogeneity.93. A nucleic acid encoding the RNA polymerase of any one of paragraphs73-92.94. A vector comprising the nucleic acid of paragraph 93.95. A host cell comprising the nucleic acid of paragraph 93 or thevector of paragraph 94.96. A kit comprising the RNA polymerase of any one of paragraphs 73-92.97. A composition comprising the RNA polymerase of any one of paragraphs73-92.98. A ribonucleic acid (RNA) produced by the method of any one ofparagraphs 89-92.99. The RNA of paragraph 98 formulated in a lipid nanoparticle.100. The RNA of paragraph 98, wherein the lipid nanoparticle comprises amolar ratio of 20-60% ionizable amino lipid, 5-25% non-cationic lipid,25-55% sterol, and 0.5-15% PEG-modified lipid.101. An RNA polymerase comprising at least one additional C-terminalamino acid relative to a corresponding wild-type RNA polymerase,optionally wherein the at least one additional C-terminal amino acidcomprises glycine (G) and/or alanine (A).102. The RNA polymerase of paragraph 101, wherein the RNA polymerase isselected from T7 RNA polymerases, T3 RNA polymerases, and SP6 RNApolymerases.103. The RNA polymerase of paragraph 101 or 102, wherein the RNApolymerase further comprises at least one additional amino acidsubstitution, optionally further comprising an amino acid substitutioncorresponding to an amino acid substitution of SEQ ID NO:1 selected fromS43A, G47A, R257A, and G259A.104. The RNA polymerase of any one of paragraphs 101-104, wherein theRNA polymerase is a T7 RNA polymerase comprising an amino acid sequenceat least 90%, at least 95%, or at least 98% identical to SEQ ID NO:1modified to include an amino acid substitution at position 43, 47, 257,and/or 259, optionally wherein the amino acid substitution is alanine(A).105. The RNA polymerase of any one of paragraphs 101-104, wherein theRNA polymerase is a T3 RNA polymerase comprising an amino acid sequenceat least 90%, at least 95%, or at least 98% identical to SEQ ID NO:6modified to include an amino acid substitution at position 44, 48, 258,and/or 260, optionally wherein the amino acid substitution is alanine(A).106. The RNA polymerase of any one of paragraphs 101-104, wherein theRNA polymerase is a SP6 RNA polymerase comprising an amino acid sequenceat least 90%, at least 95%, or at least 98% identical to SEQ ID NO:7modified to include an amino acid substitution at position 15, 19, 230,and/or 232, optionally wherein the amino acid substitution is alanine(A).107. A T7 RNA polymerase comprising an amino acid sequence at least 90%,at least 95%, or at least 98% identical to SEQ ID NO:1 modified toinclude an amino acid substitution at position 43, 47, 257, and/or 259,optionally wherein the amino acid substitution is alanine (A).108. A T3 RNA polymerase comprising an amino acid sequence at least 90%,at least 95%, or at least 98% identical to SEQ ID NO:6 modified toinclude an amino acid substitution at position 44, 48, 258, and/or 260,optionally wherein the amino acid substitution is alanine (A).109. A SP6 RNA polymerase comprising an amino acid sequence at least90%, at least 95%, or at least 98% % identical to SEQ ID NO:7 modifiedto include an amino acid substitution at position 15, 19, 230, and/or232, optionally wherein the amino acid substitution is alanine (A).

TABLE 1 RNA Polymerase Sequences RNA SEQ ID PolymeraseAmino Acid Sequence NO T7 RNAMNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 1 PolymeraseRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAT3 RNA MNIIENIEKNDFSEIELAAIPENTLADHYGSALAKEQLALEHESYELGERR 6 PolymeraseFLKMLERQAKAGEIADNAAAKPLLATLLPKLTTRIVEWLEEYASKKGRKPSAYAPLQLLKPEASAFITLKVILASLTSTNMTTIQAAAGMLGKAIEDEARFGRIRDLEAKHFKKHVEEQLNKRHGQVYKKAFMQVVEADMIGRGLLGGEAWSSWDKETTMHVGIRLIEMLIESTGLVELQRHNAGNAGSDHEALQLAQEYVDVLAKRAGALAGISPMFQPCVVPPKPWVAITGGGYWANGRRPLALVRTHSKKGLMRYEDVYMPEVYKAVNLAQNTAWKINKKVLAVVNEIVNWKNCPVADIPSLERQELPPKPDDIDTNEAALKEWKKAAAGIYRLDKARVSRRISLEFMLEQANKFASKKAIWFPYNMDWRGRVYAVPMFNPQGNDMTKGLLTLAKGKPIGEEGFYWLKIHGANCAGVDKVPFPERIAFIEKHVDDILACAKDPINNTWWAEQDSPFCFLAFCFEYAGVTHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAQKVNEILKQDAINGTPNEMITVTDKDTGEISEKLKLGTSTLAQQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLDDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWDAVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTKEILRHRCAVHWTTPDGFPVWQEYRKPLQKRLDMIFLGQFRLQPTINTLKDSGIDAHKQESGIAPNEVHSQDGSHLRMTVVYAHEKYGIESEALIHDSFGTIPADAGKLFKAVRETMVITYENNDVLADFYSQFADQLHETQLDKMPPLPK KGNLNLQDILKSDFAFASP6 RNA MQDLHAIQLQLEEEMFNGGIRRFEADQQRQIAAGSESDTAWNRRLLSELIA 7 PolymerasePMAEGIQAYKEEYEGKKGRAPRALAFLQCVENEVAAYITMKVVMDMLNTDATLQAIAMSVAERIEDQVRFSKLEGHAAKYFEKVKKSLKASRTKSYRHAHNVAVVAEKSVAEKDADFDRWEAWPKETQLQIGTTLLEILEGSVFYNGEPVFMRAMRTYGGKTIYYLQTSESVGQWISAFKEHVAQLSPAYAPCVIPPRPWRTPFNGGFHTEKVASRIRLVKGNREHVRKLTQKQMPKVYKAINALQNTQWQINKDVLAVIEEVIRLDLGYGVPSFKPLIDKENKPANPVPVEFQHLRGRELKEMLSPEQWQQFINWKGECARLYTAETKRGSKSAAVVRMVGQARKYSAFESIYFVYAMDSRSRVYVQSSTLSPQSNDLGKALLRFTEGRPVNGVEALKWFCINGANLWGWDKKTFDVRVSNVLDEEFQDMCRDIAADPLTFTQWAKADAPYEFLAWCFEYAQYLDLVDEGRADEFRTHLPVHQDGSCSGIQHYSAMLRDEVGAKAVNLKPSDAPQDIYGAVAQVVIKKNALYMDADDATTFTSGSVTLSGTELRAMASAWDSIGITRSLTKKPVMTLPYGSTRLTCRESVIDYIVDLEEKEAQKAVAEGRTANKVHPFEDDRQDYLTPGAAYNYMTALIWPSISEVVKAPIVAMKMIRQLARFAAKRNEGLMYTLPTGFILEQKIMATEMLRVRTCLMGDIKMSLQVETDIVDEAAMMGAAAPNFVHGHDASHLILTVCELVDKGVTSIAVIHDSFGTHADNTLTLRVALKGQMVAMYIDGNALQKLLEEHEVRWMVDTGIEVPEQGEFDLNEIM DSEYVFA K11 RNAMNALNIGRNDFSEIELAAIPYNILSEHYGDQAAREQLALEHEAYELGRQRF 8 PolymeraseLKMLERQVKAGEFADNAAAKPLVLTLHPQLTKRIDDWKEEQANARGKKPRAYYPIKHGVASELAVSMGAEVLKEKRGVSSEAIALLTIKVVLGNAHRPLKGHNPAVSSQLGKALEDEARFGRIREQEAAYFKKNVADQLDKRVGHVYKKAFMQVVEADMISKGMLGGDNWASWKTDEQMHVGTKLLELLIEGTGLVEMTKNKMADGSDDVTSMQMVQLAPAFVELLSKRAGALAGISPMHQPCVVPPKPWVETVGGGYWSVGRRPLALVRTHSKKALRRYADVHMPEVYKAVNLAQNTPWKVNKKVLAVVNEIVNWKHCPVGDVPAIEREELPPRPDDIDTNEVARKAWRKEAAAVYRKDKARQSRRCRCEFMVAQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGSLTLAKGKPIGLDGFYWLKIHGANCAGVDKVPFPERIKFIEENEGNILASAADPLNNTWWTQQDSPFCFLAFCFEYAGVKHHGLNYNCSLPLAFDGSCSGIQHFSAMLRDSIGGRAVNLLPSDTVQDIYKIVADKVNEVLHQHAVNGSQTVVEQIADKETGEFHEKVTLGESVLAAQWLQYGVTRKVTKRSVMTLAYGSKESLVRQQVLEDTIQPAIDNGEGLMFTHPNQAAGYMAKLIWDAVTVTVVAAVEAMNWLKSAAKLLAAEVKDKKTKEVLRKRCAIHWVTPDGFPVWQEYRKQNQARLKLVFLGQANVKMTYNTGKDSEIDAHKQESGIAPNFVHSQDGSHLRMTVVHANEVYGIDSEALIHDSSGTIPADAGNLFKAVRETMVKTYEDNDVIADFYDQFADQLHESQLDKMPAVPAKGDLNLRDILESDFAFA

TABLE 2 RNA Polymerase C-Terminal Variant Sequences RNA SEQ IDPolymerase Amino Acid Sequence NO T7 RNAMNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF  99 Polymerase C-RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA TerminalFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGR VariantIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 T7 RNAMNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 100 Polymerase C-RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA TerminalFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGR VariantIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSW (additional G)HKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGT3 RNA MNIIENIEKNDFSEIELAAIPFNTLADHYGSALAKEQLALEHESYELGERR 101Polymerase C- FLKMLERQAKAGEIADNAAAKPLLATLLPKLTTRIVEWLEEYASKKGRKPSTerminal AYAPLQLLKPEASAFITLKVILASLTSTNMTTIQAAAGMLGKAIEDEARFG VariantRIRDLEAKHFKKHVEEQLNKRHGQVYKKAFMQVVEADMIGRGLLGGEAWSSWDKETTMHVGIRLIEMLIESTGLVELQRHNAGNAGSDHEALQLAQEYVDVLAKRAGALAGISPMFQPCVVPPKPWVAITGGGYWANGRRPLALVRTHSKKGLMRYEDVYMPEVYKAVNLAQNTAWKINKKVLAVVNEIVNWKNCPVADIPSLERQELPPKPDDIDTNEAALKEWKKAAAGIYRLDKARVSRRISLEFMLEQANKFASKKAIWFPYNMDWRGRVYAVPMFNPQGNDMTKGLLTLAKGKPIGEEGFYWLKIHGANCAGVDKVPFPERIAFIEKHVDDILACAKDPINNTWWAEQDSPFCFLAFCFEYAGVTHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAQKVNEILKQDAINGTPNEMITVTDKDTGEISEKLKLGTSTLAQQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLDDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWDAVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTKEILRHRCAVHWTTPDGFPVWQEYRKPLQKRLDMIFLGQFRLQPTINTLKDSGIDAHKQESGIAPNFVHSQDGSHLRMTVVYAHEKYGIESFALIHDSFGTIPADAGKLFKAVRETMVITYENNDVLADFYSQFADQLHETQLDKMPPLPK KGNLNLQDILKSDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 T3 RNAMNIIENIEKNDFSEIELAAIPENTLADHYGSALAKEQLALEHESYELGERR 102 Polymerase C-FLKMLERQAKAGEIADNAAAKPLLATLLPKLTTRIVEWLEEYASKKGRKPS TerminalAYAPLQLLKPEASAFITLKVILASLTSTNMTTIQAAAGMLGKAIEDEARFG VariantRIRDLEAKHFKKHVEEQLNKRHGQVYKKAFMQVVEADMIGRGLLGGEAWSSWDKETTMHVGIRLIEMLIESTGLVELQRHNAGNAGSDHEALQLAQEYVDVLAKRAGALAGISPMFQPCVVPPKPWVAITGGGYWANGRRPLALVRTHSKKGLMRYEDVYMPEVYKAVNLAQNTAWKINKKVLAVVNEIVNWKNCPVADIPSLERQELPPKPDDIDTNEAALKEWKKAAAGIYRLDKARVSRRISLEFMLEQANKFASKKAIWFPYNMDWRGRVYAVPMFNPQGNDMTKGLLTLAKGKPIGEEGFYWLKIHGANCAGVDKVPFPERIAFIEKHVDDILACAKDPINNTWWAEQDSPFCFLAFCFEYAGVTHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAQKVNEILKQDAINGTPNEMITVTDKDTGEISEKLKLGTSTLAQQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLDDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWDAVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTKEILRHRCAVHWTTPDGFPVWQEYRKPLQKRLDMIFLGQFRLQPTINTLKDSGIDAHKQESGIAPNFVHSQDGSHLRMTVVYAHEKYGIESFALIHDSFGTIPADAGKLFKAVRETMVITYENNDVLADFYSQFADQLHETQLDKMPPLPK KGNLNLQDILKSDFAFAGSP6 RNA MQDLHATQLQLEEEMENGGIRRFEADQQRQIAAGSESDTAWNRRLLSELIA 103Polymerase C- PMAEGIQAYKEEYEGKKGRAPRALAFLQCVENEVAAYITMKVVMDMLNTDATerminal TLQATAMSVAERIEDQVRFSKLEGHAAKYFEKVKKSLKASRTKSYRHAHNV VariantAVVAEKSVAEKDADFDRWEAWPKETQLQIGTTLLEILEGSVFYNGEPVFMRAMRTYGGKTIYYLQTSESVGQWISAFKEHVAQLSPAYAPCVIPPRPWRTPFNGGFHTEKVASRIRLVKGNREHVRKLTQKQMPKVYKAINALQNTQWQINKDVLAVIEEVIRLDLGYGVPSFKPLIDKENKPANPVPVEFQHLRGRELKEMLSPEQWQQFINWKGECARLYTAETKRGSKSAAVVRMVGQARKYSAFESIYFVYAMDSRSRVYVQSSTLSPQSNDLGKALLRFTEGRPVNGVEALKWECINGANLWGWDKKTFDVRVSNVLDEEFQDMCRDIAADPLTFTQWAKADAPYEFLAWCFEYAQYLDLVDEGRADEFRTHLPVHQDGSCSGIQHYSAMLRDEVGAKAVNLKPSDAPQDIYGAVAQVVIKKNALYMDADDATTFTSGSVTLSGTELRAMASAWDSIGITRSLTKKPVMTLPYGSTRLTCRESVIDYIVDLEEKEAQKAVAEGRTANKVHPFEDDRQDYLTPGAAYNYMTALIWPSISEVVKAPIVAMKMIRQLARFAAKRNEGLMYTLPTGFILEQKIMATEMLRVRTCLMGDIKMSLQVETDIVDEAAMMGAAAPNFVHGHDASHLILTVCELVDKGVTSIAVIHDSFGTHADNTLTLRVALKGQMVAMYIDGNALQKLLEEHEVRWMVDTGIEVPEQGEFDLNEIM DSEYVFAX_(n), where X is any amino acid and n is anyinteger, e.g., between 1 and 5 SP6 RNAMQDLHATQLQLEEEMENGGIRRFEADQQRQIAAGSESDTAWNRRLLSELIA 104 Polymerase C-PMAEGIQAYKEEYEGKKGRAPRALAFLQCVENEVAAYITMKVVMDMLNTDA TerminalTLQATAMSVAERIEDQVRFSKLEGHAAKYFEKVKKSLKASRTKSYRHAHNV VariantAVVAEKSVAEKDADFDRWEAWPKETQLQIGTTLLEILEGSVFYNGEPVFMRAMRTYGGKTIYYLQTSESVGQWISAFKEHVAQLSPAYAPCVIPPRPWRTPFNGGFHTEKVASRIRLVKGNREHVRKLTQKQMPKVYKAINALQNTQWQINKDVLAVIEEVIRLDLGYGVPSFKPLIDKENKPANPVPVEFQHLRGRELKEMLSPEQWQQFINWKGECARLYTAETKRGSKSAAVVRMVGQARKYSAFESIYFVYAMDSRSRVYVQSSTLSPQSNDLGKALLRFTEGRPVNGVEALKWECINGANLWGWDKKTFDVRVSNVLDEEFQDMCRDIAADPLTFTQWAKADAPYEFLAWCFEYAQYLDLVDEGRADEFRTHLPVHQDGSCSGIQHYSAMLRDEVGAKAVNLKPSDAPQDIYGAVAQVVIKKNALYMDADDATTFTSGSVTLSGTELRAMASAWDSIGITRSLTKKPVMTLPYGSTRLTCRESVIDYIVDLEEKEAQKAVAEGRTANKVHPFEDDRQDYLTPGAAYNYMTALIWPSISEVVKAPIVAMKMIRQLARFAAKRNEGLMYTLPTGFILEQKIMATEMLRVRTCLMGDIKMSLQVETDIVDEAAMMGAAAPNFVHGHDASHLILTVCELVDKGVTSIAVIHDSFGTHADNTLTLRVALKGQMVAMYIDGNALQKLLEEHEVRWMVDTGIEVPEQGEFDLNEIM DSEYVFAG K11 RNAMNALNIGRNDFSEIELAAIPYNILSEHYGDQAAREQLALEHEAYELGRQRF 105 Polymerase C-LKMLERQVKAGEFADNAAAKPLVLTLHPQLTKRIDDWKEEQANARGKKPRA TerminalYYPIKHGVASELAVSMGAEVLKEKRGVSSEAIALLTIKVVLGNAHRPLKGH VariantNPAVSSQLGKALEDEARFGRIREQEAAYFKKNVADQLDKRVGHVYKKAFMQVVEADMISKGMLGGDNWASWKTDEQMHVGTKLLELLIEGTGLVEMTKNKMADGSDDVTSMQMVQLAPAFVELLSKRAGALAGISPMHQPCVVPPKPWVETVGGGYWSVGRRPLALVRTHSKKALRRYADVHMPEVYKAVNLAQNTPWKVNKKVLAVVNEIVNWKHCPVGDVPAIEREELPPRPDDIDTNEVARKAWRKEAAAVYRKDKARQSRRCRCEFMVAQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGSLTLAKGKPIGLDGFYWLKIHGANCAGVDKVPFPERIKFIEENEGNILASAADPLNNTWWTQQDSPFCFLAFCFEYAGVKHHGLNYNCSLPLAFDGSCSGIQHFSAMLRDSIGGRAVNLLPSDTVQDIYKIVADKVNEVLHQHAVNGSQTVVEQIADKETGEFHEKVTLGESVLAAQWLQYGVTRKVTKRSVMTLAYGSKESLVRQQVLEDTIQPAIDNGEGLMFTHPNQAAGYMAKLIWDAVTVTVVAAVEAMNWLKSAAKLLAAEVKDKKTKEVLRKRCAIHWVTPDGFPVWQEYRKQNQARLKLVFLGQANVKMTYNTGKDSEIDAHKQESGIAPNFVHSQDGSHLRMTVVHANEVYGIDSFALIHDSSGTIPADAGNLFKAVRETMVKTYEDNDVIADFYDQFADQLHESQLDKMPAVPAKGDLNLRDILESDFAFAX _(n), where Xis any amino acid and n is any integer, e.g., between 1 and 5 K11 RNAMNALNIGRNDFSEIELAAIPYNILSEHYGDQAAREQLALEHEAYELGRQRF 106 Polymerase C-LKMLERQVKAGEFADNAAAKPLVLTLHPQLTKRIDDWKEEQANARGKKPRA TerminalYYPIKHGVASELAVSMGAEVLKEKRGVSSEAIALLTIKVVLGNAHRPLKGH VariantNPAVSSQLGKALEDEARFGRIREQEAAYFKKNVADQLDKRVGHVYKKAFMQVVEADMISKGMLGGDNWASWKTDEQMHVGTKLLELLIEGTGLVEMTKNKMADGSDDVTSMQMVQLAPAFVELLSKRAGALAGISPMHQPCVVPPKPWVETVGGGYWSVGRRPLALVRTHSKKALRRYADVHMPEVYKAVNLAQNTPWKVNKKVLAVVNEIVNWKHCPVGDVPAIEREELPPRPDDIDTNEVARKAWRKEAAAVYRKDKARQSRRCRCEFMVAQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGSLTLAKGKPIGLDGFYWLKIHGANCAGVDKVPFPERIKFIEENEGNILASAADPLNNTWWTQQDSPFCFLAFCFEYAGVKHHGLNYNCSLPLAFDGSCSGIQHFSAMLRDSIGGRAVNLLPSDTVQDIYKIVADKVNEVLHQHAVNGSQTVVEQIADKETGEFHEKVTLGESVLAAQWLQYGVTRKVTKRSVMTLAYGSKESLVRQQVLEDTIQPAIDNGEGLMFTHPNQAAGYMAKLIWDAVTVTVVAAVEAMNWLKSAAKLLAAEVKDKKTKEVLRKRCAIHWVTPDGFPVWQEYRKQNQARLKLVFLGQANVKMTYNTGKDSEIDAHKQESGIAPNFVHSQDGSHLRMTVVHANEVYGIDSFALIHDSSGTIPADAGNLFKAVRETMVKTYEDNDVIADFYDQFADQLHESQLDKMPAVPAKGDLNLRDILESDFAFAG

EXAMPLES Example 1. Production of Variant T7 RNA Polymerases

C-helix and C-linker T7 RNA polymerase variants were generated with thesubstitutions shown in Tables 3 and 4.

TABLE 3 T7 RNA Polymerase Variants T7 RNA Polymerase SEQ ID VariantsAmino Acid Sequence NO S43A MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHE AYEMGEARF 2 RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAG47A MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEM A EARF 3RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAR257A MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 4RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIA T AAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAG259A MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 5RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIA TRA AALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAE42A MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHASYEMGEARF 9RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAE421 MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHISYEMGEARF 10RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAE42L MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHLSYEMGEARF 11RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAE42M MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHMSYEMGEARF 12RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAE42K MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHKSYEMGEARF 13RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAE42Q MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHQSYEMGEARF 14RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAS43I MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHEIYEMGEARF 15RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAS43L MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHELYEMGEARF 16RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAS43M MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHEMYEMGEARF 17RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAS43K MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHEKYEMGEARF 18RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAS43Q MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHEQYEMGEARF 19RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAS43E MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHEEYEMGEARF 20RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAY44A MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESAEMGEARF 21RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAY44I MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESIEMGEARF 22RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAY44L MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESLEMGEARF 23RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAY44M MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESMEMGEARF 24RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAY44K MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESKEMGEARF 25RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAY44Q MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESQEMGEARF 26RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAY44E MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESEEMGEARF 27RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAE45A MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYAMGEARF 28RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAE45I MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYIMGEARF 29RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAE45L MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYLMGEARF 30RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAE45M MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYMMGEARF 31RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAE45K MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYKMGEARF 32RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAE45Q MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYQMGEARF 33RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAM46A MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEAGEARF 34RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAM46I MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEIGEARF 35RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAM46L MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYELGEARF 36RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAM46K MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEKGEARF 38RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAM46Q MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEQGEARF 39RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAM46E MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEEGEARF 40RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAG47I MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMIEARF 41RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAG47L MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMLEARF 42RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAG47M MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMMEARF 43RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAG47K MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMKEARF 44RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAG47Q MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMQEARF 45RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAG47E MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMEEARF 46RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAA255I MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 315RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIITIAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAA255K MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 316RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIKTIAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAA255Q MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 317RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIQTIAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAA255Y MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 318RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIYTIAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAR257I MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 47RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATIAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAR257L MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 48RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATLAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAR257M MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 49RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATMAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAR257K MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 50RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATKAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAR257Q MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 51RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATQAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAR257E MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 52RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATEAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAA258I MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 53RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRIGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAA258L MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 54RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRLGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAA258M MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 55RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRMGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAA258K MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 56RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRKGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAA258Q MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 57RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRQGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAA258E MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 58RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATREGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAG259I MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 59RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAIALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAG259L MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 60RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRALALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEWWTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAG259M MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 61RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAMA1AGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAG259K MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 62RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAKALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAG259Q MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 63RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAQALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAG259E MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 64RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAEALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAA260I MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 65RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGILAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAA260L MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 66RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGLLAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAA260M MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 67RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGMLAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAA260K MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 68RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGKLAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAA260Q MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 69RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGQLAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAA260E MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 70RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGELAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAL261A MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 71RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGAAAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAL261I MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 72RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGAIAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAL261M MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 73RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGAMAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAL261K MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 74RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGAKAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAL261Q MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 75RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGAQAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAL261E MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 76RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGAEAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAA262I MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 77RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALIGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAA262L MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 78RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALLGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAA262M MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 79RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALMGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAA262K MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 80RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALKGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAA262Q MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 81RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALQGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAA262E MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 82RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALEGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAS43R MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHE R YEMGEARF 83RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAE45R MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESY R MGEARF 84RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAG47R MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEM R EARF 85RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAR257W MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 86RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIA T WAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAG259R MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 87RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIA TRA RALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAA260R MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 88RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIA TRAG RLAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAN165W MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 89RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGR IRDLEAKHFKK WVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAE167M MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 90RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGR IRDLEAKHFKKNV MEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAE167N MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 91RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGR IRDLEAKHFKKNV NEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAE168I MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 92RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGR IRDLEAKHFKKNVE IQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAE168T MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 93RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGR IRDLEAKHFKKNVE TQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAE168V MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 94RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGR IRDLEAKHFKKNVE VQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAA181F MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 95RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKK F FMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAA181W MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 96RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKK W FMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAQ184M MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 97RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFM M VVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAE187F MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 98RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVV F ADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFA

TABLE 4 T7 RNA Polymerase C-Terminal Variants T7 RNA Polymerase C-Terminal SEQ ID Variant Amino Acid Sequence NO S43A C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHE A YEMGEARF 107 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 S43A C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHE A YEMGEARF 108 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA Variant (S43AFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGR C-terminal GIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSW Variant orHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIA S43A*)TRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGG47A C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEM A EARF 109Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 G47A C-MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHESYEM A EARF 110 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGR (G47A C-IRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSW terminal GHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIA Variant orTRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALM G47A*)RYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGR257A C- MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHESYEMGEARF 111Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIA T AAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 R257A C-MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHESYEMGEARF 112 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIA T AAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGG259A C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 113Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIA TRA AALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 G259A C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 114 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIA TRA AALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGE42A C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHASYEMGEARF 115 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 E42A C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHASYEMGEARF 116 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA ValiantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGE42I C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHISYEMGEARF 117 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA ValiantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 E42I C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHISYEMGEARF 118 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGE42L C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHLSYEMGEARF 119 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 E42L C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHLSYEMGEARF 120 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGE42M C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHMSYEMGEARF 121 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 E42M C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHMSYEMGEARF 122 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGE42K C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHKSYEMGEARF 123 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 E42K C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHKSYEMGEARF 124 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGE42Q C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHQSYEMGEARF 125 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 E42Q C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHQSYEMGEARF 126 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGS43I C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHEIYEMGEARF 127 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 S43I C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHEIYEMGEARF 128 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGS43L C- MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHELYEMGEARF 129 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 S43L C-MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHELYEMGEARF 130 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGS43M C- MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHEMYEMGEARF 131 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 S43M C-MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHEMYEMGEARF 132 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGS43K C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHEKYEMGEARF 133 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 S43K C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHEKYEMGEARF 134 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGS43Q C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHEQYEMGEARF 135 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 S43Q C-MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHEQYEMGEARF 136 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGS43E C- MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHEEYEMGEARF 137 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 S43E C-MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHEEYEMGEARF 138 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGY44A C- MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHESAEMGEARF 139 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 Y44A C-MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHESAEMGEARF 140 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGY44I C- MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHESIEMGEARF 141 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 Y44I C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESIEMGEARF 142 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGY44L C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESLEMGEARF 143 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 Y44L C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESLEMGEARF 144 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGY44M C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESMEMGEARF 145 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 Y44M C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESMEMGEARF 146 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGY44K C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESKEMGEARF 147 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 Y44K C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESKEMGEARF 148 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGY44Q C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESQEMGEARF 149 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 Y44Q C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESQEMGEARF 150 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGY44E C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESEEMGEARF 151 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 Y44E C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESEEMGEARF 152 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGE45A C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYAMGEARF 153 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 E45A C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYAMGEARF 154 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGE45I C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYIMGEARF 155 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 E45I C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYIMGEARF 156 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGE45L C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYLMGEARF 157 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 E45L C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYLMGEARF 158 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGE45M C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYMMGEARF 159 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 E45M C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYMMGEARF 160 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGE45K C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYKMGEARF 161 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 E45K C-MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHESYKMGEARF 162 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGE45Q C- MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHESYQMGEARF 163 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 E45Q C-MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHESYQMGEARF 164 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGM46A C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEAGEARF 165 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 M46A C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEAGEARF 166 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGM46I C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEIGEARF 167 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 M46I C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEIGEARF 168 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGM46L C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYELGEARF 169 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 M46L C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYELGEARF 170 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGM46K C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEKGEARF 173 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 M46K C-MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHESYEKGEARF 174 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGM46Q C- MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHESYEQGEARF 175 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 M46Q C-MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHESYEQGEARF 176 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGM46E C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEEGEARF 177 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 M46E C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEEGEARF 178 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGG47I C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMIEARF 179 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 G47I C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMIEARF 180 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGG47L C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMLEARF 181 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 G47L C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMLEARF 182 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGG47M C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMMEARF 183 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 G47M C-MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHESYEMMEARF 184 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGG47K C- MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHESYEMKEARF 185 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 G47K C-MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHESYEMKEARF 186 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGG47Q C- MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHESYEMQEARF 187 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAXn, where X is any amino acid and n is any integer, e.g., between 1 and 5G47Q C- MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHESYEMQEARF 188 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGG47E C- MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHESYEMEEARF 189 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 G47E C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMEEARF 190 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGR257I C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 191Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATIAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 R257I C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 192 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATIAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGR257L C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 193Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATLAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 R257L C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 194 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATLAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGR257M C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 195Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATMAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 R257M C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 196 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATMAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGR257K C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 197Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATKAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 R257K C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 198 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATKAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGR257Q C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 199Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATQAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 R257Q C-MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHESYEMGEARF 200 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATQAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGR257E C- MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHESYEMGEARF 201Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATEAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 R257E C-MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHESYEMGEARF 202 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATEAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGA258I C- MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHESYEMGEARF 203Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRIGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 A258I C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 204 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRIGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGA258L C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 205Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRLGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 A258L C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 206 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRLGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGA258M C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 207Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRMGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 A258M C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 208 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRMGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGA258K C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 209Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRKGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 A258K C-MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHESYEMGEARF 210 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRKGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGA258Q C- MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHESYEMGEARF 211Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRQGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 A258Q C-MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHESYEMGEARF 212 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRQGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGA258E C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 213Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATREGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 A258E C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 214 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATREGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGG259I C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 215Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAIALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 G259I C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 216 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAIALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGG259L C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 217Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRALALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 G259L C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 218 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRALALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGG259M C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 219Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAMALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 G259M C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 220 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAMALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGG259K C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 221Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAKALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 G259K C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 222 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAKALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGG259Q C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 223Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAQALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 G259Q C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 224 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAQALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGG259E C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 225Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAEALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 G259E C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 226 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAEALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGA260I C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 227Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGILAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 A260I C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 228 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGILAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGA260L C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 229Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGLLAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 A260L C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 230 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGLLAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGA260M C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 231Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGMLAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 A260M C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 232 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGMLAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGA260K C- MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHESYEMGEARF 233Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGKLAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 A260K C-MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHESYEMGEARF 234 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGKLAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGA260Q C- MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHESYEMGEARF 235Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGQLAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 A260Q C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 236 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGQLAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGA260E C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 237Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGELAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 A260E C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 238 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGELAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGL261A C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF  37Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGAAAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 L261A C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 239 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGAAAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGL261I C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 240Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGAIAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 L261I C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 241 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGAIAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGL261M C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 242Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGAMAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 L261M C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 243 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGAMAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGL261K C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 244Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGAKAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 L261K C-MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHESYEMGEARF 245 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGAKAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGL261Q C- MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHESYEMGEARF 246Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGAQAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 L261Q C-MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHESYEMGEARF 247 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGAQAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGL261E C- MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHESYEMGEARF 248Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGAEAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 L261E C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 249 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGAEAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGA262I C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 250Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALIGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 A262I C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 251 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALIGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGA262L C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 252Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALLGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 A262L C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 253 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALLGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGA262M C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 254Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALMGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 A262M C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 255 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALMGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGA262K C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 256Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALKGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 A262K C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 257 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALKGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGA262Q C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 258Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALQGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 A262Q C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 259 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALQGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGA262E C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 260Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALEGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 A262E C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 261 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALEGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGS43R C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHE R YEMGEARF 262Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 S43R C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHE R YEMGEARF 263 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGE45R C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESY R MGEARF 264Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 E45R C-MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHESY R MGEARF 265 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGG47R C- MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHESYEM R EARF 266Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 G47R C-MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHESYEM R EARF 267 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGR257W C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 268terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA variantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIA T WAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 R257W C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 269 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIA T WAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGG259R C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 270Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIA TRA RALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 G259R C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 271 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIA TRA RALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGA260R C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 272Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIA TRAG RLAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 A260R C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 273 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIA TRAG RLAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGN165W C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 274Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGR IRDLEAKHFKK WVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 N165W C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 275 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGR IRDLEAKHFKK WVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKG NLNLRDILESDFAFAGE167M C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 276Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGR IRDLEAKHFKKNV MEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 E167M C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 277 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGR IRDLEAKHFKKNV MEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGE167N C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 278Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGR IRDLEAKHFKKNV NEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 E167N C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 279 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGR IRDLEAKHFKKNV NEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGE168I C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 280Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGR IRDLEAKHFKKNVE IQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 E168I C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 281 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGR IRDLEAKHFKKNVE IQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGE168T C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 282Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGR IRDLEAKHFKKNVE TQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 E168T C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 283 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGR IRDLEAKHFKKNVE TQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGE168V C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 284Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGR IRDLEAKHFKKNVE VQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 E168V C-MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHESYEMGEARF 285 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGR IRDLEAKHFKKNVE VQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGA181F C- MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHESYEMGEARF 286Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKK F FMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 A181F C-MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHESYEMGEARF 287 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKK F FMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGA181W C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 288Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKK W FMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 A181W C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 289 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKK W FMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGQ184M C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 290Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFM M VVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 Q184M C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 291 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFM M VVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGE187F C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 292Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVV F ADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAX_(n), where X is any amino acid and nis any integer, e.g., between 1 and 5 E187F C-MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 293 TerminalRKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA VariantFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVV F ADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGT7 RNA MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 294Polymerase C- RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTATerminal FQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGR Variant (C-IRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSW terminal GG)HKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGGT7 RNA MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 295Polymerase C- RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTATerminal (C- FQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRterminal A) IRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAAT7 RNA MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARF 296Polymerase C- RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTATerminal FQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGR Variant (C-IRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSW terminal AA)HKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAAAG47A C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEM A EARF 297Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA Variant (C-FQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGR terminal GG)IRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAGGG47A C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEM A EARF 298Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA Variant (C-FQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGR terminal A)IRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAAG47A C- MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEM A EARF 299Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA Variant (C-FQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGR terminal AA)IRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAAAS43A/G47A MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHEAYEM A EARF 300RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAS43A/G47A C- MNTINIAKNDFSDIELAAIPENTLADHYGERLAREQLALEHEAYEM A EARF 301Terminal RKMFERQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTA Variant (C-FQFLQEIKPEAVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGR terminal G)IRDLEAKHFKKNVEEQLNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIESTGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWTGITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKDKARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPECFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRDEVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEKVKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAHKQESGIAPNEVHSQDGSHLRKTVVWAHEKYGIESEALIHDSFGTIPADAANLFKAVRETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAK GNLNLRDILESDFAFAG

Example 2. The Purity of hEPO mRNA Produced Using the T7 RNA PolymeraseVariants is Comparable to the Purity of hEPO mRNA Produced Using aWild-Type T7 RNA Polymerase

In vitro transcription reactions were performed using hEPO DNA templateand (1) Wild-type (WT) T7 RNA polymerase (WT #1 and WT #2), (2) a G47AC-terminal G T7 RNA polymerase variant (“G47A*”) (SEQ ID NO: 110), and(3) a S43A C-terminal G T7 RNA polymerase variant (“S43A*”) (SEQ ID NO:108). DBAA (dibutylammonium acetate) HPLC analyses showed that thepurity of all hEPO transcripts was approximately the same (FIG. 1).

Example 3. hEPO mRNA Produced Using the T7 RNA Polymerase Variants doesnot Elicit a Cellular Cytokine Response

BJ fibroblasts were transfected with hEPO mRNA produced by the IVTreactions described in Example 2. The mRNA was either unpurified orpurified by reverse phase (RP) chromatography. As shown in FIG. 2 (leftgraph), unpurified mRNA produced using the S43A* T7 RNA polymerasevariant or the G47A* T7 RNA polymerase variant was as ‘cold’ (did notelicit a cytokine response) as purified mRNA produced using WT T7polymerase. Approximately equivalent mRNA expression levels are shown inFIG. 2 (right graph). The mRNA used in these experiments did not containany nucleotide modifications.

A similar cell transfection assay was repeated using hEPO mRNAcontaining either m¹ψ nucleotide modifications (FIG. 3, top graph) ormo⁵U nucleotide modifications (FIG. 3, bottom graph). BJ fibroblastcells transfected with unpurified m¹ψ hEPO mRNA produced using the G47A*and S43A* T7 RNA polymerase variants exhibited lower IFNβ responses thancells transfected with unpurified m¹ψ hEPO mRNA produced using WT T7 RNApolymerase. Approximately equivalent mRNA expression levels are shown inFIG. 4.

Monocyte-derived macrophages (MDMs) were then used to examine cytokineresponse further. An IP10 to hypoxanthine-guaninephosophoribosyltransferase (HPRT) ratio was measured in MDMs transfectedwith hEPO mRNA generated under the conditions described in Example 2.MDMs are very sensitive to the unmodified chemistry, so drasticdifferences between samples were not seen using unmodified hEPO mRNA.However, MDMs transfected with unpurified m¹ψ hEPO mRNA produced usingthe G47A* and S43A* T7 RNA polymerase variants exhibited lower IP10responses than MDMs transfected with purified m¹ψ hEPO mRNA producedusing WT T7 RNA polymerase (FIG. 5, top graph).

Example 4. The T7 RNA Polymerase Variants Produce mRNA Associated withLess Contaminating dsRNA than the WT T7 RNA Polymerase

A standard dsRNA ELISA was used to assess dsRNA contaminants (e.g.,longer than 40 nucleotide base pairs) in IVT reactions used to produceunmodified hEPO mRNA (FIG. 7) or hEPO mRNA containing either m¹ψnucleotide modifications (FIG. 8, top graph) or mo⁵U nucleotidemodifications (FIG. 8, bottom graph). The dsRNA assay demonstrated thatthe S43A* T7 RNA polymerase variant and the G47A* T7 RNA polymerasevariant produced less dsRNA than WT T7 RNA polymerase (FIGS. 7 and 8).

Example 5. The T7 RNA Polymerase Variants Reduce 3′ Heterogeneity ofmRNA

The 3′ heterogeneity of transcripts can be measured using a RNAse T1digest. RNAse T1 cleaves mRNA specifically after a G nucleotide.Endonucleolytic cleavage results in a 5′ hydroxide (OH) and 3′monophosphate (mP) ‘scar’. Thus, a RNAse T1 digest can be used todifferentiate between transcripts that do and do not have non-templatedadditions at the 3′ end. In this Example, the hEPO mRNA produced inExample 2 terminate with a polyA tail and a XbaI restriction site (e.g.A_(n)UCUAG). hEPO mRNA produced using WT T7 RNA polymerase, the S43A* T7RNA polymerase variant, or the G47A* T7 RNA polymerase variant weredigested with RNAse T1 and analyzed by LCMS to generate oligofingerprints. A clean 3′ end (5′ OH/3′ OH) was found to peak at 32780Da, while a ‘scar’ (5′ OH/3′ mP) peaks at 32860 Da (FIG. 9A). A ‘scar’indicates that the transcript had non-templated additions at the carboxyterminus. hEPO mRNA produced using the T7 RNA polymerase variants, S43A*or G47A*, had a higher 3′ end population distribution, indicating thatthey have cleaner 3′ ends and improved 3′ end heterogeneity (FIG. 9B).

Example 6. T7 RNA Polymerase Variants Produce ‘Cleaner’ 3′ Ends

An assay was used to probe the extent of non-templated additionsproduced by T7 RNAP variants, G47A* and S43A*. In this assay, ligationleftmers were made using an enzymatic polymerase (see, e.g., FIG. 11A).The leftmers were then annealed to a fully-complimentary DNA splintimmediately adjacent to a 5′-monophosporylated rightmer RNA. A DNAsplint is typically 40 nt long and the rightmer is typically decoratedwith a fluorophore at the 5′-end. DNA ligase I was then added undercatalytic conditions and ligation was allowed to proceed for a desiredreaction time before the reaction was quenched with EDTA. The mixturewas then denatured in 4M urea at 95° C. for 5 minutes before beingloaded onto a denaturing polyacrylamide gel (PAGE-D) (FIG. 11B). Thepercentage of acrylamide in the gel depends on the length of theexpected construct but is typically 6% acrylamide for leftmers >50 nt inlength and 20% acrylamide for leftmers approaching the length of typicalmRNA. 6% acrylamide gels ran for 25 minutes at a constant 180V and 20%acrylamide gels ran for 120 minutes at a constant 180V. The gels werethen imaged on a Typhoon or similar scanner using excitation andemission wavelengths appropriate to the fluorophore present on therightmer and the newly ligated product. Rightmer and ligated productsmigrate differently on the gel based upon size, and the intensity of thetwo bands will exactly correspond to the efficiently of the ligationreaction. The greater the extent of untemplated nucleotide additionduring the enzymatic synthesis of the leftmer, the lower the expectedligation yield.

Ligation yield was calculated as a ratio of bands corresponding tounligated rightmer and ligated full-length product. The reaction wasterminated early to exaggerate differences in reaction yields. Based onthese results T7 RNAP variant G47A* incorporated the fewest untemplatednucleotides at the 3′-end of the leftmer RNA.

Example 7. Production of Capped mRNAs in a Single In Vitro TranscriptionReaction

In a standard in vitro transcription (IVT) assay, T7 RNA polymeraseprefers to initiate transcription with a 5′GTP. A single stranded RNA(ssRNA) molecule produced by such standard IVT assays requires aseparate enzymatic capping step (FIG. 13). For example, a cap1 RNA(7mGpppN2′-Om-RNA) can be produced in a capping assay using a Vacciniacapping enzyme and a 2′-O-methyl transferase (2′OMTase). Cap1 istypically used for efficient protein translation and mRNA stability incells. However, some mRNA sequences are difficult to cap, andcapping/methylation enzymes are very expensive.

The present disclosure provides, in some embodiments, methods of cappinga ssRNA (e.g., mRNA) with trinucleotides co-transcriptionally in an invitro transcription assay using a T7 RNA polymerase variant describedherein (e.g., T7 RNA polymerase variant G47A*). Efficientco-transcriptional capping typically includes a double-stranded DNA(dsDNA) template that initiates transcription with 5′ATP and equimolarconcentrations of NTPs and trinucleotides. Normally, T7 RNA polymerasehas severely reduced initiation activity with 5′ATP. However, in thepresence of XAG (wherein X is any nucleotide, e.g., 5′^(m)Gppp)trinucleotides, the limited initiation activity of T7 RNA polymerasewith 5′ATP drives initiation with the trinucleotide rather than 5′ATP,generating capped mRNA products co-transcriptionally.

Exemplary commercially available di- and trinucleotide caps are shown inFIG. 14A. The Vaccinia cap1 is a typical di-nucleotide cap and cannot beadded co-transcriptionally. Another di-nucleotide cap that can be addedco-transcriptionally is the Anti-Reverse Cap Analog (ARCA), alsocommercially available (e.g., from Thermo Fisher, Catalog number:AM8045). ARCA is a modified cap analog in which the 3′ OH group (closerto m⁷G) is replaced with —OCH3. ARCA is used as a control in some of theco-transcriptional capping assays described in this study.

In a co-transcriptional capping assay, human EPO (hEPO) DNA template wasused. Also present in the assay mixture were 5 mM NTP with or without 5mM trinucleotides (^(m)GpppAG, ^(m)GmpppAG, and ARCA). The unmodifiedmRNA products were analyzed by RNase H cap assay or LCMS. As shown inTable 5, both wild type (WT) T7 RNA polymerase and the T7 RNA polymerasevariant G47A* were able to produce fully capped mRNA with equally highefficiency. RNA yield was comparable among different IVT reactions (FIG.14B) and mRNA products with high degree of integrity were produced (FIG.14C).

TABLE 5 Capping Efficiency of WT T7 RNA polymerase or T7 RNA polymerasevariant G47A* T7 RNA polymerase Trinucleotide Capping efficiency (%) WT— 0.0 T7 RNA polymerase variant — 0.0 G47A* WT m⁷GpppAG 93.9 T7 RNApolymerase variant m⁷GpppAG 100.0 G47A* WT m⁷GpppAG 100.0 T7 RNApolymerase variant m⁷GpppAG 100.0 G47A*

The immune-stimulatory activity of the mRNA products was assessed byevaluating their ability to induce cytokine (IFNβ) production in BJfibroblasts. Interestingly, WT T7 RNA polymerase produced unmodifiedmRNA that induced a high cytokine response, while T7 RNA polymerasevariant G47A* produced unmodified mRNA that did not stimulate cytokineproduction in BJ fibroblasts (FIG. 14D), minimizing the need to furtherpurify the RNA after the IVT/capping assay. hEPO expression from themRNA products were also evaluated. No hEPO expression was observed frommRNA produced by WT T7 RNA polymerase, while mRNA produced by T7 RNApolymerase variant G47A* led to comparable hEPO expression as mRNAmodified with 1-methyl-pseudourine capped with Vaccinia cap1 (FIG. 14E).LCMS experiments also shows that T7 RNA polymerase variant G47A*produced cleaner RNA than WT T7 RNA polymerase, though the trinucleotideincorporation efficiency appeared to be equivalent for the two enzymes(FIG. 15).

In a different experiment, the capping efficiency of T7 RNA polymerasevariant G47A* was compared with the efficiency achieved in a standardcapping assay using the Vaccinia cap1. In this experiment, the reactionmixture contained a hEPO dsDNA template with a deoxythymidine atposition +1 on the template strand (also termed “Astart” for the firsttemplated nucleotide), the T7 RNA polymerase variant G47A*, andequimolar (7.5 mM) NTPs and GAG trinucleotides. The resulting mRNAproducts were purified using oligo dT and analyzed for cappingefficiency, in vitro cytokine response, and in vitro expression. Theresults show that T7 RNA polymerase variant G47A* produced capped mRNAwith comparably high efficiency as the standard capping process (Table6), that the mRNA did not induce cytokine response in BJ fibroblasts(FIG. 16A), and that the mRNA lead to high hEPO expression in BJfibroblasts (FIG. 16B).

TABLE 6 Capping Efficiency of Co-transcriptional Capping and StandardProcess Cap identity % functional cap Uncap 0 GAG (one-pot) 96.0Vaccinia cap1 (+RP) 99.8

In summary, it was demonstrated herein that the T7 RNA polymerasevariant G47A* can efficiently initiate transcription at the 3′dTTP ofthe template strand in the presence of XAG trinucleotides (e.g., GAG orGmAG). Provided at equimolar concentrations as the NTPs in the assays,the trinucleotides can be added co-transcriptionally to produce fullycapped mRNAs that does not stimulate an immune response and results inhigh protein expression (FIG. 17).

Example 8. Comparison of Transcription Initiation with 5′GTP (Gstart) or5′ATP (Astart)

Using the co-transcriptional capping assays described in Example 7, thecapping efficiency on model constructs where transcription initiationrequires 5′-GTP (Gstart) or a 5′-ATP (Astart) were compared. A modelconstruct encoding a 5′UTR and a 47-mer RNA oligonucleotide was used astemplate for IVT. The total RNA products were assessed by massspectrometry and the results show that Astart constructs moreefficiently incorporate the GAG or GmAG trinucleotides, compared toGstart constructs (FIG. 18A). Though the data is not shown here, it wasalso observed that Cstart and Ustart constructs produced low amounts ofcapped products.

Further, the RNA products were subject to RNase H cleavage and thecleaved 5′ ends of the RNA products were analyzed using massspectrometry to determine the presence of the cap. Similar results wereobtained as that of the total RNA products (FIG. 18B).

A trinucleotide titration experiment was also performed using the modelconstruct used in FIGS. 18A and 18B. In the co-transcriptional cappingassay, 5 mM of NTPs (A, modified U, G, C at equimolar ratio) were usedwhile the concentration of the trinucleotide (GAG or GmAG) varied toevaluate capping efficiency at different molar ratios of the NTP and thetrinucleotide. The crude RNA products were then analyzed by LC-MS. Theresult shows that capping efficiency is the highest when the NTP and thetrinucleotide are at equimolar ratio for both GAG and GmAG (Table 7).

TABLE 7 Trinucleotide Titration Trinucleotide concentration (mM) % Cap(GAG) % Cap (GmAG) 5 (EQ) 96.0 88.3 4 92.8 85.6 3 90.3 82.8 2 81.7 74.51 66.1 57.0 0.5 42.5 48.6 0 0 0

Example 9. Capping of mRNAs Chemically Modified with1-Methyl-Pseudouridine

Co-transcriptional capping of mRNAs chemically modified with1-methyl-pseudouridine were also assessed. Three model constructs wereused: hEPO, Luc (encoding luciferase), and eGFP. All IVT templates wereAstart templates. The templates may be PCR fragments or plasmids. Thecapping assay reaction mixture contained the template, T7 RNA polymerasevariant G47A*, 7.5 mM of NTPs, and 7.5 mM of trinucleotides (GAG orGmAG). The UTPs in the NTPs were replaced by 1-methylpseudouridine toproduce chemically-modified mRNA. Vaccinia cap1 was used as a productioncontrol in a standard capping assay. The mRNA products were analyzedusing RNase H cap assay, T1 fingerprinting, and a fragment analyzer todetermine their integrity. The abilities of the mRNA products ininducing cytokine response and expressing the encoded protein in BJfibroblasts were also tested.

As shown in Table 8, the mRNA chemically modified with1-methyl-pseudouridine for all three model constructs produced from PCRfragment templates were efficiently capped, the capping efficiency wascomparable to the vaccinia cap1 control. Chemistry A denotes use ofunmodified uridine triphosphate nucleotide and Chemistry B denotes useof 1-methyl-pseudouridine triphosphate nucleotide. Chemistry C denotes5-methyl-cytidine triphosphate and 1-methyl-pseudouridine triphosphatenucleotides. Similar results were obtained for mRNA chemically modifiedwith 1-methyl-pseudouridine produced from plasmid templates (Table 9).Further, RNase T1 fingerprinting analysis showed that the mRNAs producedfrom the model constructs using PCR fragment templates were of correctsequence (FIG. 19A). The mRNA produced from either PCR fragmenttemplates (FIG. 19B) or plasmid templates (FIG. 20C) were also shown tohave high degree of integrity. The capped mRNAs produced from PCRfragment templates as well as plasmid templates did not induce anycytokine expression in BJ fibroblasts (FIG. 19C, FIG. 20D). Further,mRNAs produced from plasmid templates resulted in expression of theencoded proteins in BJ fibroblasts (FIGS. 20E-20G). The expressionlevels for GAG-capped mRNAs were slightly higher, comparable or slightlylower than the expression levels from the Vaccinia cap1 control mRNAs.

TABLE 8 Capping Efficiency of mRNA chemically modified with 1-methyl-pseudouridine - PCR Fragment Templates Construct Cap % Cap (% reported)hEPO Uncap  0 hEPO Cap1 (vaccinia) 99.8 (n/a) hEPO GAG (trinucleotide)96.1 hEPO GmAG (trinucleotide) 94.9 Luc Uncap  0 Luc Cap1 (vaccinia)85.7 (100%) Luc GAG (trinucleotide) 94.4 Luc GmAG (trinucleotide) 93.3eGFP Uncap  0 eGFP Cap1 (vaccinia) 99.4 (88%) eGFP GAG (trinucleotide)93.8 eGFP GmAG (trinucleotide) 94.9

TABLE 9 Capping Efficiency of mRNA chemically modified with 1-methyl-pseudouridine - Plasmid Templates Construct Cap % Cap (% reported) hEPOUncap  0 hEPO Cap1 (vaccinia) 99.8 (n/a) hEPO GAG (trinucleotide) 96.4hEPO GmAG (trinucleotide) 96.3 Luc Uncap  0 Luc Cap1 (vaccinia) 85.7(100%) Luc GAG (trinucleotide) 95.7 Luc GmAG (trinucleotide) 99.6eGFPegd Uncap  0 eGFPdeg Cap1 (vaccinia) 92.1 (n/a) eGFPdeg GAG(trinucleotide) 94.3 eGFPdeg GmAG (trinucleotide) 92.1

The NTP consumption in the co-transcriptional capping assay were alsoassessed for reactions using plasmid templates for model constructs hEPO(FIG. 20A) and eGFP (FIG. 20B). The results show that the trinucleotidesGAG and GmAG were hardly consumed throughout the duration of the assay(2 hours). This is consistent with the fact that the concentration ofthe trinucleotides in the assay was in extreme excess. It may bepossible to recover trinucleotides after the assay is completed.

Example 10. Substitutions at the T7 RNA Polymerase C-Terminus Impact RNAYields

G47A* polymerase contains an extra glycine at the C-terminus (“footglycine”) of the translated protein. The presence of this foot glycinewas confirmed by trypsin digest and mass spectrometry analysis ofpurified G47A* protein (data not shown). The role of the C-terminal footregion of G47A* was examined in greater detail because of its proximityto the active site of G47A*.

In vitro transcription (IVT) reactions were performed using hEPO DNAtemplate and (1) a wild-type (WT) T7 RNA polymerase (WT), (2) a WT T7RNA polymerase with a foot glycine (WT+G), (3) a WT T7 RNA polymerasewith two foot glycines (WT+GG), (4) a wild-type RNA polymerase with twofoot alanines (WT+AA), (5) a G47A T7 RNA polymerase variant, (6) a G47A*T7 RNA polymerase variant with a foot glycine (G47A+G), (7) a G47A T7RNA polymerase variant with two foot glycines (G47A+GG), (7) a G47A T7RNA polymerase variant with two foot alanines (G47A+AA), (9) a S43A/G47AT7 RNA polymerase variant (S43A/G47A), and (10) a S43A/G47A T7 RNApolymerase variant with a foot glycine (S43A/G47A+G). IVT reactionmixtures contained hEPO DNA template, one of the T7 RNA polymerasevariants listed above, and NTPs. IVT reactions with unmodified chemistry(FIG. 22) were performed with standard NTPs (ATP, CTP, GTP, UTP), whileIVT reactions with pseudouridine-modified (m1ψ) chemistry (FIG. 23)contained 1-methylpseudouridine instead of UTP to producechemically-modified mRNA. RNA yield was measured by UV absorption.

hEPO mRNA yield decreased with the presence of a foot glycine in eitherWT or G47A* variant T7 RNA polymerase (FIGS. 22-23). mRNA yieldcontinued to decrease with either two foot glycines or two foot alanineresidues, although there was less of a decrease with G47A* than with WTT7 RNA polymerase.

Example 11. Impact of T7 RNA Polymerase Foot Glycine on 3′-mRNAHeterogeneity

hEPO mRNA produced using WT or G47A* T7 RNAP with a foot glycine wasdigested with RNAse T1 and analyzed by LCMS to generate oligofingerprints as in Example 5. WT T7 RNAP IVT reactions were conductedwith either equimolar concentrations of NTPs (WT EQ) or with molarexcess of GTP and ATP (WT alpha (A)). LCMS analysis revealed a clean 3′end (5′ OH/3′ OH) peak at 32780 Da, and a ‘scar’ (5′ OH/3′ mP) peak at32860 Da (FIG. 24A). A ‘scar’ indicates that the transcript hadnon-templated additions at the 3′ end. hEPO mRNA produced using WT T7RNAP and equimolar concentrations of NTPs (WT EQ) had decreased 3′ endpopulation distribution, indicating decreased 3′ homogeneity and morerun-on products with non-templated additions (FIG. 24B). However, hEPOmRNA produced using G47A* T7 RNAP with a foot glycine had a higher 3′end population distribution, indicating that they have cleaner 3′ endsand improved 3′ end heterogeneity (FIG. 24B).

To directly examine the effect of the T7 RNAP foot region on hEPO 3′mRNA homogeneity, IVT reactions were performed using hEPO DNA templateand (1) a wild-type T7 RNAP (WT), (2) a WT T7 RNAP with a foot glycine(WT+G), (3) a WT T7 RNAP with two foot glycines (WT+GG), (4) a wild-typeT7 RNAP with two foot alanines (WT+AA), (5) a G47A T7 RNAP variant, (6)a G47A* T7 RNAP variant with a foot glycine (G47A+G), (7) a G47A T7 RNAPvariant with two foot glycines (G47A+GG), (8) a G47A T7 RNAP variantwith two foot alanines (G47A+AA), (9) a S43A/G47A T7 RNAP variant(S43A/G47A), and (10) a S43A/G47A T7 RNAP variant with a foot glycine(S43A/G47A+G). IVT reaction mixtures contained hEPO DNA template, one ofthe T7 RNAP variants listed above, and equimolar NTPs. IVT reactionswere conducted which produced either unmodified mRNA (FIG. 24C) orpseudouridine modified mRNA (FIG. 24D). The hEPO mRNA was digested withRNase T1 as in Example 5 and above. The presence of a foot glycineincreased hEPO mRNA 3′ end homogeneity by 60% (FIGS. 24C-24D). Two footglycines or two foot alanines in T7 RNAP also improved 3′ endhomogeneity by 60-70% over WT or G47A T7 RNAPs (FIGS. 24C-24D). Theseresults indicate the presence of a single foot glycine in T7 RNAP canincrease the 3′ end homogeneity observed in FIG. 24B.

Example 12. Impact of T7 RNAP Foot Glycine on Immune Stimulation

The role of T7 RNAP foot glycine in stimulating an immune response tohEPO mRNA products was assessed by evaluating their ability to inducecytokine (IFNβ) production in BJ fibroblasts as in Example 7.Interestingly, while WT T7 RNAP-produced unmodified andpseudouridine-modified hEPO mRNA induced a high cytokine response, WT T7RNAP with a foot glycine-produced unmodified and pseudouridine-modifiedhEPO mRNA stimulated less cytokine production in BJ fibroblasts (FIG.25). hEPO mRNA produced by G47A* variant T7 RNAP with a foot glycinefailed to stimulate cytokine production in BJ fibroblasts, indicating anadditive effect between the foot glycine and G47A T7 RNA polymerasevariant in failing to induce an immune response (FIG. 25).

Example 13. Impact of T7 RNA Polymerase Foot Glycine on mRNA Production

An assay was developed to probe the effect of a foot glycine residue onhEPO mRNA species produced by WT and G47A variant T7 RNAPs. The T7 RNAPenzymes tested were: (1) WT+G (foot glycine), (2) G47A variant, or (3)G47A variant+G (G47A* variant). IVT reactions contained one of the T7RNAPs listed, shORFan template, NTPs (either unmodified or modified UTP)and ³²P-CTP. Reactions were terminated with EDTA after the desiredamount of time. The reaction mixture was then denatured in 4M urea at95° C. for 5 minutes before being loaded onto a denaturingpolyacrylamide gel (20% acrylamide) (FIG. 26A). The gel ran for 30 minat 20 watts and then an additional 2 h at 40 watts. The image wastransferred onto a phosphorimager screen (exposure time of 1 h) prior toimaging using the Typhoon scanner. Use of ³²P-CTP labels full-lengthproduct (arrow denoting shORFan mRNA), run-on transcription products(box above) (FIG. 26A), and reverse complement mRNA products (FIG. 26A,lower box). The results are also summarized in Table 10 below. Thesedata demonstrate that T7 RNAP with a foot glycine is associated withless contaminating dsRNA than either WT or G47A T7 RNAP (FIG. 26A (lowerbox) and FIG. 26B). Furthermore, a foot glycine is associated withdecreased run-on transcription (FIG. 26A (upper box), FIG. 26B) comparedwith WT or G47A variant T7 RNAP.

TABLE 10 Ratio reverse complement (RC): full length (FL) mRNAs Fulllength Mutant RC:FL (compared to G47A + G) WT + G (Unmodified mRNA) —0.3 G47A (Unmodified mRNA) 0.16 1 G47A + G (G47A*) (Unmodified — 1 mRNA)WT + G (Pseudouridine mRNA) — 0.2 G47A (Pseudouridine mRNA) 0.18 1.2G47A + G (Pseudouridine mRNA) — 1

Example 14. Evaluation of Repeat Dosing of Trinuc-Capped G47A* mRNAEncoding Firefly Luciferase

In vitro transcribed mRNA is typically purified, for example, by reversephase chromatography (RP) to remove cytokine-inducing impurities (dsRNA)and process impurities (e.g., protein/DNA), and to improve total RNApurity. This purification process, however, often results in asignificant loss of mRNA product Eliminating RP reducesturn-around-time, eliminates operational and engineering complexity atscale, and is cost saving. Further, because the high temperatures usedduring RP can hydrolyze a fraction of mRNA, eliminating RP increasesmRNA expression levels. Thus, we have developed a mRNA production andpurification strategy to eliminate the need for RP. The mRNAs producedby the methods provided in this Example are referred to as“trinuc-capped G47A* mRNAs,” which are mRNAs capped with thetrinucleotide GpppA_(2′Ome)pG co-transcriptionally in an in vitrotranscription assay using the G47A* T7 RNAP variant (having a G47Asubstitution and an additional C-terminal G).

In this Example, a single 0.5 mpk dose of mRNA encoding fireflyluciferase (ffLuc) was formulated in MC3 lipid nanoparticles andadministered weekly to C57Bl/6 mice (n=5) for 6 weeks (on Day 1, Day 8,Day 15, Day 22, Day 29, and Day 36). The following mRNAs wereadministered: (1) unmodified mRNA produced using wild-type T7 polymerasein the presence of equimolar concentrations of NTPs and purified byoligo dT purification (Chemistry A Process 1 dT); (2)pseudouridine-modified mRNA produced using wild-type T7 polymerase inthe presence of an excess concentration of GTP/ATP, and purified byreverse phase chromatography (Chemistry B Process 2 RP); (3)pseudouridine-modified mRNA produced using wild-type T7 polymerase inthe presence of an excess concentration of GTP/ATP and purified by oligodT purification (Chemistry B Process 2 dT); (4) pseudouridine-modifiedmRNA produced using wild-type T7 polymerase in the presence of equimolarconcentrations of NTPs and purified by oligo dT purification (ChemistryB process 1 dT); (5) pseudouridine-modified trinuc-capped G47A* mRNA(trinucleotide capped and produced using the G47A* T7 RNAP variant)purified by reverse phase chromatography (Chemistry B Process 3 RP); (6)pseudouridine-modified trinuc-capped G47A* mRNA purified by oligo dTpurification (Chemistry B Process 3 dT); and (7) uncappedpseudouridine-modified mRNA produced using the G47A* T7 RNAP variant andpurified by oligo dT purification (Chemistry B Process 4 dT)). Six hoursfollowing each dose, the following were assessed: serum cytokine levels,mRNA expression, and anti-PEG IgM levels. B cell activation was assessedsix hours following the Day 36 dose. Chemistry A denotes use ofunmodified uridine triphosphate nucleotide and Chemistry B denotes useof 1-methyl-pseudouridine triphosphate nucleotide. Process 1 refers toequimolar NTPs in the IVT and vaccinia cap1, Process 2 refers to an IVTcontaining 4:2:1:1 GTP:ATP:CTP:UTP (see WO 2018/053209 A1, published 22Mar. 2018, incorporated herein by reference in its entirety) andvaccinia cap1, Process 3 refers to equimolar NTPs and GAG in the IVT,and Process 4 refers to equimolar NTPs in the IVT and no cap.

Results from the serum cytokine assessment (IP-10) in mice at Day 1 andDay 29 are presented in FIG. 27A and FIG. 27B, showing thattrinuc-capped G47A* mRNAs induce serum cytokine levels in vivo, similarto alpha mRNA controls. The results were similar for in vitroexperiments in which the mRNAs were delivered to BJ human fibroblasts(FIG. 28A) and monocyte-derived macrophages (MDMs) (FIG. 28B).

The mRNA expression studies (FIG. 29) showed that trinuc-capped G47A*mRNAs maintained high expression in vivo following 6 weekly doses.

The anti-PEG IgM assessment (FIG. 30) showed that N1U trinuc-cappedG47A* mRNAs produce low anti-PEG IgM levels following 6 weekly doses.

The B cell activation analysis (FIG. 31) showed that activation ofsplenic B cells transfected with trinuc-capped G47A* mRNAs was low,similar to the alpha mRNA controls.

Collectively, these results indicate that trinuc-capped G47A* mRNAsencoding ffLuc, which are not subjected to RP purification, do notinduce a cytokine response above baseline, maintain high expressionlevels in vivo, maintain low anti-PEG IgM levels in vivo, and exhibitlow B cell activation.

Example 15. Evaluation of Repeat Dosing of Trinuc-Capped G47A* mRNAEncoding Human Erythropoietin

In this Example, a single 0.5 mpk dose of mRNA encoding humanerythropoietin (hEPO) was formulated in MC3 lipid nanoparticles andadministered weekly to C57Bl/6 mice (n=5) for 6 weeks (on Day 1, Day 8,Day 15, Day 22, Day 29, and Day 36). The following mRNAs wereadministered: (1) unmodified mRNA produced using wild-type T7 polymerasein the presence of equimolar concentrations of NTPs and purified byoligo dT purification (Chemistry A Process 1 dT); (2)pseudouridine-modified mRNA produced using wild-type T7 polymerase inthe presence of an excess concentration of GTP/ATP and purified byreverse phase chromatography (Chemistry B Process 2 RP); (3)pseudouridine-modified mRNA produced using wild-type T7 polymerase inthe presence of an excess concentration of GTP/ATP and purified by oligodT purification (Chemistry B Process 2 dT); (4) pseudouridine-modifiedmRNA produced using wild-type T7 polymerase in the presence of equimolarconcentrations of NTPs and purified by oligo dT purification (ChemistryB Process 1 dT); (5) pseudouridine-modified trinuc-capped G47A* mRNA(trinucleotide capped and produced using the G47A* T7 RNAP variant)purified by reverse phase chromatography (Chemistry B Process 3 RP); (6)pseudouridine-modified trinuc-capped G47A* mRNA purified by oligo dTpurification (Chemistry B Process 3 dT); and (7) uncappedpseudouridine-modified mRNA produced using the G47A* T7 RNAP variant andpurified by oligo dT purification (Chemistry B Process 4 dT)). Six hoursfollowing each dose, the following were assessed: serum cytokine levels,mRNA expression, and anti-PEG IgM levels. B cell activation was assessedsix hours following the Day 36 dose. Chemistry A denotes use ofunmodified uridine triphosphate nucleotide and Chemistry B denotes useof 1-methyl-pseudouridine triphosphate nucleotide. Process 1 refers toequimolar NTPs in the IVT and vaccinia cap1, Process 2 refers to an IVTcontaining 4:2:1:1 GTP:ATP:CTP:UTP and vaccinia cap1, Process 3 refersto equimolar NTPs and GAG in the IVT, and Process 4 refers to equimolarNTPs in the IVT and no cap.

Results from the serum cytokine assessment (IP-10) in mice at Day 1 andDay 22 are presented in FIG. 32A and FIG. 32B, showing thattrinuc-capped G47A* mRNAs induce serum cytokine levels in vivo, similarto alpha mRNA controls. The results were similar for in vitroexperiments in which the mRNAs were delivered to BJ human fibroblasts(FIG. 33A) and monocyte-derived macrophages (MDMs) (FIG. 33B).

The mRNA expression studies (FIG. 34) showed that trinuc-capped G47A*mRNAs maintained high expression in vivo following 6 weekly doses.

The anti-PEG IgM assessment (FIG. 35) showed that trinuc-capped G47A*mRNAs produce low anti-PEG IgM levels following 6 weekly doses.

The B cell activation analysis (FIG. 36) showed that activation ofsplenic B cells transfected with trinuc-capped G47A* mRNAs was low,similar to the alpha mRNA controls.

Collectively, these results indicate that trinuc-capped G47A* mRNAsencoding hEPO, which are not subjected to RP purification, do not inducea cytokine response above baseline, maintain high expression levels invivo, maintain low anti-PEG IgM levels in vivo, and exhibit low B cellactivation.

Example 16. Evaluation of Indel Frequency and Point Mutation Frequency

mRNAs were prepared with either WT or G47A* enzymes using Process 1(equimolar NTPs) or Process 2 (4:2:1:1 GTP:ATP:CTP:UTP). For nextgeneration sequencing (NGS), the mRNAs were converted to cDNA by reversetranscriptase and adaptors were ligated to prepare the sequencinglibrary. The NGS data was compared to the parent sequence and any indelsobserved were tabulated. The indel frequency was comparable for WT andG47A* (FIG. 37A). Likewise, the NGS data was analyzed for any pointmutations. The point mutation frequency was comparable for WT and G47A*(FIG. 37B).

All references, patents and patent applications disclosed herein areincorporated by reference with respect to the subject matter for whicheach is cited, which in some cases may encompass the entirety of thedocument.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

1. A T7 ribonucleic acid (RNA) polymerase variant comprising an aminoacid sequence having at least 90% identity to the amino acid sequence ofSEQ ID NO: 1 modified to comprise an amino acid substitution at positionG47, wherein the amino acid substitution causes a loop structure of theT7 RNA polymerase variant to undergo a conformational change to a helixstructure as the T7 RNA polymerase variant transitions from aninitiation complex to an elongation complex. 2.-6. (canceled)
 7. The RNApolymerase variant of claim 1, wherein the amino acid substitution isselected from the group consisting of alanine, isoleucine, leucine,methionine, lysine, glutamine, and glutamate.
 8. The RNA polymerasevariant of claim 7, wherein the amino acid substitution is alanine(G47A).
 9. The RNA polymerase variant of claim 1, wherein the T7 RNApolymerase variant comprises an amino acid sequence having identity toSEQ ID NO:1. 10.-12. (canceled)
 13. The RNA polymerase variant of claim1 further comprising an additional C-terminal amino acid.
 14. The RNApolymerase variant of claim 13, wherein the additional C-terminal aminoacid comprises glycine (G).
 15. (canceled)
 16. The RNA polymerasevariant of claim 1, wherein the RNA polymerase variant comprises theamino acid sequence of SEQ ID NO:3 SEQ ID NO:110.
 17. A ribonucleic acid(RNA) polymerase variant comprising an additional C-terminal amino acidrelative to wild-type RNA polymerase. 18.-20. (canceled)
 21. The RNApolymerase variant of claim 14 comprising two additional C-terminalamino acids.
 22. The RNA polymerase variant of claim 21, wherein the twoadditional C-terminal amino acids comprise the same type of amino acidor two different types of amino acids. 23.-32. (canceled)
 33. A methodof producing a ribonucleic acid (RNA) comprising contacting a DNAtemplate with the T7 RNA polymerase of claim 1 under conditions thatresult in the production of RNA transcript.
 34. A method of performingan in vitro transcription (IVT) reaction, comprising contacting a DNAtemplate with the RNA polymerase of claim 1 in the presence ofnucleoside triphosphates and buffer under conditions that result in theproduction of RNA transcript.
 35. The method of claim 34, wherein: theRNA transcript produced, when delivered to cells, optionally inunpurified form, stimulates a cytokine response that is at least 50%lower relative to RNA produced using wild-type RNA polymerase; theconcentration of double-stranded RNA (dsRNA) transcript produced is atleast 50% lower relative to dsRNA transcript produced using wild-typepolymerase; less than 20% of the RNA transcripts produced exhibit 3′heterogeneity; less than 50% of the RNA transcript produced isdouble-stranded contaminant; less than 50% of the RNA transcriptproduced is run-on RNA transcript; the amount of full-length RNAtranscript produced is at least 15 times greater than the amount of theDNA template; the ratio of double-stranded contaminant:full-length RNAtranscript produced is less than 1:1; and/or the RNA transcript producedhas less than 1 mutation per 100 nucleotides relative to the DNAtemplate. 36.-48. (canceled)
 49. A co-transcriptional capping method forribonucleic acid (RNA) synthesis, the method comprising reacting apolynucleotide template with the T7 RNA polymerase variant of claim 1,nucleoside triphosphates, and a cap analog under in vitro transcriptionreaction conditions to produce RNA transcript. 50.-52. (canceled) 53.The method of claim 49, wherein the nucleoside triphosphates and capanalog are present in the reaction at equimolar concentrations.
 54. Themethod of claim 49, wherein a molar ratio of cap analog to nucleosidetriphosphates, optionally wherein the nucleoside triphosphates compriseGTP and/or ATP, in the reaction is greater than 1:1 or equal to 1:1. 55.The method of claim 49, wherein the cap analog is a dinucleotide cap, atrinucleotide cap, or a tetranucleotide cap.
 56. The method of claim 55,wherein the cap is a trinucleotide cap comprising a sequence selectedfrom the following sequences: GAA, GAC, GAG, GAU, GCA, GCC, GCG, GCU,GGA, GGC, GGG, GGU, GUA, GUC, GUG, and GUU.
 57. The method of claim 56,wherein the trinucleotide cap comprises a sequence selected from thefollowing sequences: (a) m⁷GpppApA, m⁷GpppApC, m⁷GpppApG, m⁷GpppApU,m⁷GpppCpA, m⁷GpppCpC, m⁷GpppCpG, m⁷GpppCpU, m⁷GpppGpA, m⁷GpppGpC,m⁷GpppGpG, m⁷GpppGpU, m⁷GpppUpA, m⁷GpppUpC, m⁷GpppUpG, and m⁷GpppUpU;(b) m⁷G_(3′OMe)pppApA, m⁷G_(3′OMe)pppApC, m⁷G_(3′OMe)pppApG,m⁷G_(3′OMe)pppApU, m⁷G_(3′OMe)pppCpA, m⁷G_(3′OMe)pppCpC,m⁷G_(3′OMe)pppCpG, m⁷G_(3′OMe)pppCpU, m⁷G_(3′OMe)pppGpA,m⁷G_(3′OMe)pppGpC, m⁷G_(3′OMe)pppGpG, m⁷G_(3′OMe)pppGpU,m⁷G_(3′OMe)pppUpA, m⁷G_(3′OMe)pppUpC, m⁷G_(3′OMe)pppUpG, andm⁷G_(3′OMe)pppUpU; (c) m⁷G_(3′OMe)pppA_(2′OMe)pA,m⁷G_(3′OMe)pppA_(2′OMe)pC, m⁷G_(3′OMe)pppA_(2′OMe)pG,m⁷G_(3′OMe)pppA_(2′OMe)pU, m⁷G_(3′OMe)pppC_(2′OMe)pA,m⁷G_(3′OMe)pppC_(2′OMe)pC, m⁷G_(3′OMe)pppC_(2′OMe)pG,m⁷G_(3′OMe)pppC_(2′OMe)pU, m⁷G_(3′OMe)pppG_(2′OMe)pA,m⁷G_(3′OMe)pppG_(2′OMe)pC, m⁷G_(3′OMe)pppG_(2′OMe)pG,m⁷G_(3′OMe)pppG_(2′OMe)pU, m⁷G_(3′OMe)pppU_(2′OMe)pA,m⁷G_(3′OMe)pppU_(2′OMe)pC, m⁷G_(3′OMe)pppU_(2′OMe)pG, andm⁷G_(3′OMe)pppU_(2′OMe)pU; or (d) m⁷GpppA_(2′OMe)pA, m⁷GpppA_(2′OMe)pC,m⁷GpppA_(2′OMe)pG, m⁷GpppA_(2′OMe)pU, m⁷GpppC_(2′OMe)pA,m⁷GpppC_(2′OMe)pC, m⁷GpppC_(2′OMe)pG, m⁷GpppC_(2′OMe)pU,m⁷GpppG_(2′OMe)pA, m⁷GpppG_(2′OMe)pC, m⁷GpppG_(2′OMe)pG,m⁷GpppG_(2′OMe)pU, m⁷GpppU_(2′OMe)pA, m⁷GpppU_(2′OMe)pC,m⁷GpppU_(2′OMe)pG, and m⁷GpppU_(2′OMe)pU.
 58. (canceled)
 59. The methodof claim 56, wherein the trinucleotide cap comprises sequence GAG. 60.The method of claim 57, wherein the trinucleotide cap comprisesGpppA_(2′OMe)pG.
 61. The method of claim 49, wherein the polynucleotidetemplate includes a 2′-deoxythymidine residue at template position +1 ora 2′-deoxycytidine residue at template position +1. 62.-71. (canceled)72. A T7 ribonucleic acid (RNA) polymerase variant comprising an aminoacid sequence having at least 95% identity to the amino acid sequence ofSEQ ID NO: 1 modified to comprise an alanine at position
 47. 73.-77.(canceled)
 78. The T7 RNA polymerase variant of claim 72 comprising theamino acid sequence of SEQ ID NO:
 3. 79. The T7 RNA polymerase variantof claim 72 further comprising a C-terminal glycine.
 80. The T7 RNApolymerase variant of claim 79 comprising the amino acid sequence of SEQID NO:
 110. 81. A T7 RNA polymerase variant comprising the amino acidsequence of SEQ ID NO: 110.